Methods and compositions for the production of oligodendrocyte progenitor cells

ABSTRACT

Provided herein are pluripotent stem cells comprising particular combinations of transcription factor for the production of oligodendrocyte progenitor cells (OPCs). Also provided herein are methods of producing the pluripotent stem cells and methods of using the pluripotent stem cells to produce (OPCs) and oligodendrocytes.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 62/593,560, filed Dec. 1, 2017 and U.S. provisional application No. 62/624,000, filed Jan. 30, 2018, each of which is incorporated by reference herein in its entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under MH103910-01, HG008525, and AG048056 awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Oligodendrocytes are a subtype of glial cells in the central nervous system that originate from oligodendrocyte progenitor cells (OPCs). OPCs account for about 5% of cells in the central nervous system. Oligodendrocytes help support and insulate axons by producing myelin. Myelin sheaths in the central nervous system are made of extended oligodendrocyte plasma membranes. While mature oligodendrocytes cannot self-renew, OPCs can repopulate oligodendrocytes following injury to the central nervous system in healthy individuals.

Multiple sclerosis is a demyelinating disorder that includes an autoimmune response, which leads to the destruction of (damage to) the myelin sheath made by oligodendrocytes. Overactivated T-cells are thought to be part of this process, leading to a pro-inflammatory profile that results in CNS inflammation. T cells secrete various inflammatory responses such as cytokines (e.g., IFNγ, IL17, etc.), nitric oxide (NO), and glutamate, causing an overstimulation of the immune system and eventual degradation of myelin, oligodendrocytes and axons. Most treatments for demyelinating disorders have focused on reducing symptoms rather than replacing damaged myelin.

SUMMARY

Provided herein, in some embodiments, are pluripotent stem cells and methods for producing oligodendrocyte progenitor cells (OPCs) (e.g., O4-positive OPCs) and myelin-producing oligodendrocytes. In some embodiments, the oligodendrocytes are ‘immunoprotective,’ meaning they are capable of secreting immunosuppressive factors. Although myelin damage is a hallmark of many demyelinating disorders, treatment options for these disorders are limited due in part to the lack of efficient methods for producing functional OPCs. Further, while cell therapies utilizing transplanted stem cell-derived OPCs may help with remyelination, these exogenous cells can become the target of a heightened inflammatory response, resulting in some instances in limited therapeutic benefit and insignificant improvement in clinical outcome.

The experimental results provided herein show unexpectedly that particular combinations of transcription factors can induce the formation of OPCs, in some embodiments, in as few as four days without optimizing for cell growth and/or differentiation culture conditions. In some embodiments, these stem cell-derived OPCs are also engineered to protect themselves from autoimmune attacks by secreting anti-inflammatory cytokines.

Further, in some embodiments, the methods provided herein improve OPC production yield and shorten production time through the use of a PIGGYBAC™ transposon system, which permits high copy number (e.g., 5 to 50 copies) integration of nucleic acids into a host genome.

Accordingly, some aspects of the present disclosure provide pluripotent stem cells (e.g., induced pluripotent stem cells) that include at least three transcription factors selected from the following transcription factors: (a) an oligodendrocyte (OLIG) transcription factor, (b) an SRY-box (SOX) transcription factor, (c) two NKX homeobox transcription factors, and (d) an octamer-binding (OCT) transcription factor. In some embodiments, the OLIG transcription factor is OLIG2. In some embodiments, the SOX transcription factor is SOX9. In some embodiments, the two NKX homeobox transcription factor are NKX6.1 and NKX6.2. In some embodiments, the OCT transcription factor is OCT4 (also referred to as POU5F1 (POU domain, class 5, transcription factor)). In some embodiments, the pluripotent stem cells comprise, consist of, or consist essentially of OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4. In some embodiments, the pluripotent stem cells further include IL-10 and/or IFNβ.

Other aspects of the present disclosure provide methods that include introducing into pluripotent stem cells at least one engineered nucleic acid encoding at least three transcription factors selected from an OLIG transcription factor, a SOX transcription factor, two NKX homeobox transcription factors, and an OCT transcription factor to produce O4 positive OPCs. In some embodiments, the methods further include introducing into pluripotent stem cells at least one engineered nucleic acid encoding IL-10 and/or IFNβ. In some embodiments, the methods further include culturing the OPCs to produce oligodendrocytes.

Yet other aspects of the present disclosure provide pharmaceutical compositions comprising the OPCs or the oligodendrocytes produced by the methods described herein. These pharmaceutical compositions may be used, for example, to treat (e.g., improve) a demyelinating disorder, such as multiple sclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic protocol for directed differentiation of induced pluripotent stem cells (iPSCs) into oligodendrocyte progenitor cells (OPCs).

FIG. 2 shows data indicating that induction of OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4 transcription factor expression changes the morphology of iPSCs and promotes the differentiation of iPSC cells. The left picture shows the morphology of control iPSCs, in which there is no induction of transcription factor expression (no doxycycline is added to the media). The right picture shows the morphology of iPSCs after doxycycline is added to the cell media to induce expression of the transcription factors. Both pictures were taken after iPSCs were cultured for 4 days (with or without doxycycline). Scale bar=20 μm.

FIGS. 3A-3C show data indicating that induction of OLIG2, SOX9, NKX6.1, and OCT4 transcription factor expression in iPSCs promotes the formation of OPCs and maturation oligodendrocytes. FIG. 3A is a photo showing immunohistochemistry analysis of cells four days after induction of transcription factor expression, using anti-O4 antibody. FIG. 3B is a photo showing immunohistochemistry analysis of cells eight days after induction of transcription factor expression, using anti-O4 antibody. FIG. 3C is a photo showing immunohistochemistry analysis of cells eight days after induction of transcription factor expression, using anti-MOG antibody.

FIGS. 4A-4B includes data indicating that the induced OPCs are capable of forming myelin-producing cells. FIG. 4A is a bar graph showing the frequency of O4-positive, NG2-positive, and anti-Galactocerebroside (anti-GalC)-positive OPCs four days after no induction (No Dox) or after induction (Dox) of OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4 transcription factor expression in iPSCs. Data was generated using anti-O4, anti-NG2, and anti-GalC antibodies and fluorescence activated cell sorting (FACS) analysis. FIG. 4B includes transmission electron microscopy images showing robust myelin formation after engineered oligodendrocyte cells were co-cultured for 4 weeks with doxycycline inducible iNGN (Neurogenin-mediated) stem cells. Scale bars on the left two panels indicate 100 nm. Scale bars on the right two panels indicate 200 nm. M indicates myelin, while A indicates axon.

FIG. 5 shows a schematic of the process of making micro-grooves for analysis of the functional ability of OPCs. It shows in vitro myelin formation and photo-micropatterning of microgrooves for co-culture of oligodendrocyte/neuron-generating iPSCs.

FIG. 6 shows data characterizing cells with the combination of OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4 transcription factors (OPC0) and cells with a subset of the TF combination (OPC1-OPC10) using FACS analysis. The top graph shows quantification of O4 expressing iPSC-derived OPCs at Day 4 (% O4+ cells). Each column is the representation of the mean O4 expression of each independent replicate after 4 days of differentiation in mTeSR media. NoDox (left columns) indicates No doxycycline-induction and Dox (right columns) indicates doxycycline induction. (p<0.05). The bottom chart shows the combination of transcription factors tested in each cell line.

FIGS. 7A-7B include data characterizing the frequency of O4 expressing (O4+) cells in colonies from OPC0 and OPC10 cells. In each graph, the first bar for each colony indicates the frequency of marker-positive cells from cells that were un-induced (no doxycycline treatment, so cells were not induced to express the TFs). The second bar for each colony indicates the frequency of marker-positive cells from cells that were induced to express the TFs (doxycycline-treated cells). FIG. 7A shows the frequency of O4+ cells in individual colonies that were analyzed from OPC0 and OPC10. Data has not been shown for all the tested samples (n>6). Due to the large volume of colonies screened, no replicates were included. See also FIG. 14G for additional replicates and colonies. FIG. 7B shows the results for the OPC0 colony with the highest O4 marker frequency (Colony #1), which was stained for early (NG2), intermediate proliferative premitotic (O4), and late, non-proliferative post mitotic (GalC) markers after 4 or 8 days of doxycycline induction (suffix -4 or -8 respectively). The first set of bars indicates results with no doxycycline. The second set of bars for each colony indicates results with doxycycline.

FIGS. 8A-8C include RNA-seq data of O4+ cells (from OPC9) compared to uninduced samples. FIG. 8A is a heatmap showing that some of the specific oligodendrocyte markers were up-regulated after 4 days of induction confirming the commitment of the O4+ OPCs to become matured oligodendrocytes. Genes were plotted based on their log 2 fold change compared to hiPSCs and were considered as statistically significant (*) if the adjusted p-value was less than 0.05. The darker the box for each transcription factor, the higher the fold change in gene expression. Dox− indicates the absence of doxycycline treatment (TF expression was not induced). DOX+ indicates addition of doxycycline to induce TF expression. FIG. 8B is a graph showing a comparison of the mean of normalized counts and Log 2 fold change. FIG. 8C is a chart showing the top 40 differentially expressed genes, some of which are oligodendrocyte-specific such as OLIG1 and PLP1.

FIG. 9 is a schematic of an example workflow for the production of oligodendrocyte progenitor cells with increased immune-protection.

FIG. 10 is a graph of the concentration of secreted IL-10 in cells engineered to overexpress doxycycline-dependent OPC-inducing transcription factors (SOX9, NKX6.1, OLIG2, and OCT4) and doxycycline-dependent IL10 over the course of seven days. Cells were incubated in the presence or absence of doxycycline.

FIG. 11 is a graph of the concentration of secreted IL-10 in cells engineered to overexpress doxycycline-dependent OPC-inducing transcription factors and doxycycline-dependent IFNβ over the course of seven days. Cells were incubated in the presence or absence of doxycycline.

FIG. 12 is a graph of the concentration of secreted IL-10 in cells engineered to overexpress doxycycline-dependent OPC-inducing transcription factors, doxycycline-dependent IL-10, and doxycycline-dependent IFNβ over the course of seven days. Cells were incubated in the presence or absence of doxycycline.

FIGS. 13A-13B include diagrams of vectors for over-expressing IL10 and IFNβ in an orthogonal PIGGYBAC™ vector. FIG. 13A shows a vector for over-expressing IL10 and IFNβ with GFP selection. FIG. 13B shows a vector for over-expressing IL10 and IFNβ with puromycin (Puro) selection.

FIGS. 14A-14G differentiation efficiencies of colonies isolated from iPSCs transfected with OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4 (OPC0). FIG. 14A shows percent of O4-positive cells among the different colonies made by transfecting high amounts of DNA (e.g., >1000 ng per 1 million cells). The first set of bars for each colony indicates results with no doxycycline. The second set of bars for each colony indicates results with doxycycline for four days. FIG. 14B shows percent of GalC-positive cells among the different colonies made by transfecting high amounts of DNA. The first set of bars for each colony indicates results with no doxycycline. The second set of bars for each colony indicates results with doxycycline for four days. FIG. 14C shows number of genomically integrated TFs in three colonies made by transfecting high amounts of DNA. This figure shows the total copy number of all integrated transcription factors in the indicated colony. FIG. 14D shows detailed analysis of O4 and GalC expression among the top three colonies made by transfecting high amounts of DNA. FIG. 14E shows number of genomically integrated TFs among colonies made by transfecting low amounts of DNA (e.g., less than 1000 ng per 1 million cells). FIG. 14F shows detailed analysis of O4 and GalC expression in a colony made by transfecting low amounts of DNA. FIG. 14G shows percent of cells expressing NG2, 04 and GalC among colonies made by transfecting low amounts of DNA.

FIGS. 15A-15B show that the engineered OPCs could form myelin sheath in vitro. Cells from OPC0, colony 1, which were engineered to inducibly express OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4, were used. FIG. 15A shows a 3-day co-culture of engineered OPCs interacting with neurites of hiPSC-induced neurons. As shown, OPC is contacting the induced neuron. FIG. 15B shows myelin (M) formation wrapping around an axon (A), demonstrating functionality of the differentiated OPCs after four weeks of co-culture. A portion of the culture was cross-sectioned and imaged using electron microscopy.

FIGS. 16A-16C show that engineered and differentiated OPCs can myelinate human cerebral organoids. Cells from OPC0, colony 1, which were engineered to inducibly express OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4, were used. FIG. 16A shows that control cerebral organoid mixed with inducible OPCs but not treated with doxycycline did not stain for MBP or MOG. DAPI was used to label nuclei. FIG. 16B shows that cerebral organoids mixed with inducible OPCs and treated with doxycycline to induce differentiation stained positive for MBP and MOG. DAPI was used to label nuclei. FIG. 16C shows that cerebral organoids mixed with inducible OPCs and treated with doxycycline to induce differentiation stained positive for MBP. DAPI was used to label nuclei. MBP staining can be seen as the lighter shading surrounding the center two cells.

FIGS. 17A-17C include brain slice images stained for MBP demonstrating the amount of myelin (darker staining). FIG. 17A shows staining of a sample from a normal mouse. FIG. 17B shows staining of a sample from a mouse injected with engineered OPCs. Cells from OPC0, colony 1, which were engineered to inducibly express OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4, were used. FIG. 17C shows a staining of a sample from an untreated Shiverer mouse.

FIGS. 18A-18D include data quantifying the number of myelinated axons using transmission electron microscopy (TEM) images. FIGS. 18A-18B show TEM images of brain slices from control group where the mice received PBS injections (FIG. 18A) and from a treatment cohort where the mice received cells (FIG. 18B). FIG. 18C shows quantification of the number of myelinated axons in a series of brain slices. FIG. 18D shows G-ratio in injected mice.

FIGS. 19A-19B include ELISA time course data quantifying IL10 secretion from cells engineered to differentiate into oligodendrocyte progenitors (OPC0: SOX9, NKX6.1, OLIG2, NKX6.2, and OCT4) and to secrete cytokines (IL-10, or IL-10 and IFNβ). Cells were incubated in the presence or absence of doxycycline. FIG. 19A is a graph of the concentration of secreted IL-10 in cells engineered to overexpress doxycycline-dependent OPC-inducing transcription factors (SOX9, NKX6.1, OLIG2, NKX6.2, and OCT4) and doxycycline-dependent IL10. FIG. 19B shows a graph of the concentration of secreted IL-10 in cells engineered to overexpress doxycycline-dependent OPC-inducing transcription factors, doxycycline-dependent IL-10, and doxycycline-dependent IFNβ. (first series of columns: un-induced, second series of columns: induced)

FIG. 20 is a histogram showing OCT4 expression at the single cell level of cells from OPC0, colony 1, which were engineered to inducibly express OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4. The rightmost peak are cells that have not been treated with doxycycline and therefore remain OCT4-positive stem cells. The leftmost peak and peak with no OCT4 expression (shaded) are cells treated with doxycycline after four days.

FIGS. 21A-21B show adoptive transfer EAE mouse model with or without intravenous injection of hiPSC-derived OPCs engineered to secrete both IL10 and IFNβ. FIG. 21A shows the body weight over time, as days after immunization with anti-MOG T cells. Lighter line represents animals injected with cells. Black line represents animals not injected with cells. Error bars are standard errors of the mean. FIG. 21B shows the EAE clinical score. Lighter line represents animals injected with cells. Black line represents animals not injected with cells. Error bars are standard errors of the mean.

DETAILED DESCRIPTION

Induced pluripotent stem cells (iPSCs) are reprogrammed from adult differentiated cells and are capable of developing into many phenotypes. iPSCs may be obtained from a patient and changed into any cell type that is necessary to improve a particular condition, permitting patient-specific autologous clinical applications while simultaneously minimizing the risk of immune response or rejection. Although the use of stem cell therapies for clinical applications such as neurodegenerative and myelin degenerative diseases, myocardial infarction, and bone defect repair have been promising, there are significant limitations relating to uncontrolled proliferation, low cell survival, negative immune responses, differentiation into undesired cell types, inconsistency and long procedure duration. The technology provided herein overcomes many of these limitations. The present disclosure provides methods for transcription factor (TF)-mediated direct reprogramming of stem cells, such as iPSCs, to generate a desired cell type, in some instances, in as few as 1 to 8 days with high efficiency (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells expressing markers of differentiation). For example, cells may be reprogrammed within 2-8, 3-8, or 4-8 days. In some embodiments, a cell may be reprogrammed using the methods described herein in less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day. In some embodiments cells may be reprogrammed (e.g., express differentiation markers) within 1 day. The present disclosure also provides methods for protecting the desired cell type from the immune (inflammatory) response.

Autoimmune diseases that are the result of dysfunctional immune systems may be amenable to stem cell therapies. Multiple sclerosis (MS), with a worldwide prevalence of approximately 2.5 million people, is an autoimmune disease that is a frequent mesenchymal stem cell (MSC) therapy target. Success to date has been limited, however, largely due to the above-mentioned limitations. Current strategies to overcome the hurdles of stem cell are still largely impractical in application due to the lengthy culture times, complicated culture conditions and low efficiency.

Data provided herein demonstrates that TF-mediated direct reprogramming of iPSCs can be utilized, in some embodiments, to generate O4-positive OPCs while requiring no media regimen optimization. This approach is considerably faster and more efficient than existing reprogramming methods. Data provided herein also shows that these iPSCs and the cells produced from these iPSCs can be programmed to secrete inflammatory cytokines, thus rendering the cells immunoprotective.

The present disclosure is based, at least in part, on unexpected results demonstrating that a specific combination of transcription factors can induce the formation of OPCs. The combination of transcription factors, in some embodiments, comprises at least two (e.g., at least three, least four, or at least five) transcription factors selected from oligodendrocyte (OLIG) transcription factors, SRY-box (SOX) transcription factors, NKX homeobox transcription factors, and octamer-binding (OCT) transcription factors. These OPCs may be used, for example, to rebuild the damaged myelin sheath surrounding axons in subjects having demyelinating disorders.

Oligodendrocyte Progenitor Cells and Oligodendrocytes

Oligodendrocyte progenitor cells (OPCs) are a subtype of glial cells in the central nervous system characterized by expression of the proteoglycans PDGFRA and NG2 (CSPG4). They are precursors to oligodendrocytes, which are neuroglia that function to support and insulate axons by producing a myelin sheath wrapping. While mature oligodendrocytes cannot self-renew, OPCs can repopulate oligodendrocytes following an injury to the central nervous system in healthy individuals.

As stem cells develop into oligodendrocytes, each stage of development may be characterized by specific cell surface markers. For example, the membrane chondroitin sulfate proteoglycan NG2 (CSPG4) may be used as a marker of early-stage proliferative OPCs. Oligodendrocyte marker O4 may be used as an indicator of mid- to late-stage OPCs (Jackman, N et al., Physiology (24):290-7 (2009). In some embodiments, OPCs produced by the methods of the present disclosure are mid- to late-stage OPCs that express O4. Myelin Basic Protein (MBP) and Myelin Oligodendrocyte Glycoprotein (MOG) are expressed in terminal differentiation of OPCs to oligodendrocytes. They both are oligodendrocyte-specific genes and may be used as markers of mature oligodendrocytes formation. MOG is a membrane protein found on the surface of oligodendrocyte cells and on the outer layer of myelin sheaths. GalC is a galactosphingolipid of myelin found on oligodendrocyte membranes and may be used as a marker for late stage OPC (post-mitotic) and early mature oligodendrocyte. GalC may be used as a marker of terminal differentiation. In some embodiments, oligodendrocytes produced by the methods of the present disclosure may be mature oligodendrocytes that express MOG and/or GalC. Additional markers of the stages of oligodendrocyte development (e.g., markers for early-stage OPCs, intermediate and late-stage OPCs and mature oligodendrocytes) are known in the art and may be used as provided herein. See e.g., Jackman, N et al., Physiology (24):290-7 (2009).

In some embodiments, a OPC comprises an OLIG2 transcription factor and/or a nucleic acid encoding OLIG2. In some embodiments, a OPC comprises an SOX9 transcription factor and/or a nucleic acid encoding SOX9. In some embodiments, a OPC comprises an NKX6.1 transcription factor and/or a nucleic acid encoding NKX6.1. In some embodiments, a OPC comprises an NKX6.2 transcription factor and/or a nucleic acid encoding NKX6.2. In some embodiments, a OPC comprises an OCT4 transcription factor and/or a nucleic acid encoding OCT4.

In some embodiments, a OPC comprises OLIG2 and SOX9. In some embodiments, a OPC comprises OLIG2 and NKX6.1. In some embodiments, a OPC comprises OLIG2 and NKX6.2. In some embodiments, a OPC comprises OLIG2 and OCT4. In some embodiments, a OPC comprises SOX9 and NKX6.1. In some embodiments, a OPC comprises SOX9 and NKX6.2. In some embodiments, a OPC comprises SOX9 and OCT4. In some embodiments, a OPC comprises NKX6.1 and NKX6.2. In some embodiments, a OPC comprises NKX6.1 and OCT4. In some embodiments, a OPC comprises NKX6.2 and OCT4.

In some embodiments, a OPC comprises OLIG2, SOX9 and NKX6.1. In some embodiments, a OPC comprises OLIG2, SOX9 and NKX6.2. In some embodiments, a OPC comprises OLIG2, SOX9 and OCT4. In some embodiments, a OPC comprises OLIG2, NKX6.1 and NKX6.2. In some embodiments, a OPC comprises OLIG2, NKX6.1 and OCT4. In some embodiments, a OPC comprises OLIG2, NKX6.2 and OCT4. In some embodiments, a OPC comprises SOX9, NKX6.1 and NKX6.2. In some embodiments, a OPC comprises SOX9, NKX6.1 and OCT4. In some embodiments, a OPC comprises NKX6.1, NKX6.2 and OCT.

In some embodiments, a OPC comprises at least two transcription factors selected from OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4. In some embodiments, a OPC comprises at least three transcription factors selected from OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4. In some embodiments, a OPC comprises at least four transcription factors selected from OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4. In some embodiments, a OPC comprises OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4.

Pluripotent Stem Cells

Provided herein are methods for reprogramming pluripotent stem cells to produce OPCs (e.g., O4-positive OPCs). Pluripotent stem cells are cells that have the capacity to self-renew by dividing, and to develop into the three primary germ cell layers of the early embryo, and therefore into all cells of the adult body, but not extra-embryonic tissues such as the placenta. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent stem cells. ESCs are derived from the undifferentiated inner mass cells of a human embryo and are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. iPCSs can be generated directly from adult cells (Takahashi, K; Yamanaka, S. Cell 126(4):663-76, 2006). In some embodiments, a pluripotent stem cell is an ESC. In some embodiments, a pluripotent cell is an iPSC. In some embodiments, a pluripotent stem cell is a human ESC. In some embodiments, a pluripotent cell is an iPSC. In some embodiments, a pluripotent cell is a human iPSC.

Pluripotent stem cells, such as an iPSC, may be engineered to express the following transcription factors: (a) an oligodendrocyte (OLIG) transcription factor, (b) an SRY-box (SOX) transcription factor, (c) two NKX homeobox transcription factors, and (d) an octamer-binding (OCT) transcription factor. In some embodiments, a pluripotent stem cell, such as an iPSC, is engineered to express a combination of two or more transcription factors selected from OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4. In some embodiments, a pluripotent stem cell, such as an iPSC, is engineered to express OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4.

In some embodiments, a pluripotent stem cell comprises an OLIG2 transcription factor and/or a nucleic acid encoding OLIG2. In some embodiments, a pluripotent stem cell comprises an SOX9 transcription factor and/or a nucleic acid encoding SOX9. In some embodiments, a pluripotent stem cell comprises an NKX6.1 transcription factor and/or a nucleic acid encoding NKX6.1. In some embodiments, a pluripotent stem cell comprises an NKX6.2 transcription factor and/or a nucleic acid encoding NKX6.2. In some embodiments, a pluripotent stem cell comprises an OCT4 transcription factor and/or a nucleic acid encoding OCT4.

In some embodiments, a pluripotent stem cell comprises OLIG2 and SOX9. In some embodiments, a pluripotent stem cell comprises OLIG2 and NKX6.1. In some embodiments, a pluripotent stem cell comprises OLIG2 and NKX6.2. In some embodiments, a pluripotent stem cell comprises OLIG2 and OCT4. In some embodiments, a pluripotent stem cell comprises SOX9 and NKX6.1. In some embodiments, a pluripotent stem cell comprises SOX9 and NKX6.2. In some embodiments, a pluripotent stem cell comprises SOX9 and OCT4. In some embodiments, a pluripotent stem cell comprises NKX6.1 and NKX6.2. In some embodiments, a pluripotent stem cell comprises NKX6.1 and OCT4. In some embodiments, a pluripotent stem cell comprises NKX6.2 and OCT4.

In some embodiments, a pluripotent stem cell comprises OLIG2, SOX9 and NKX6.1. In some embodiments, a pluripotent stem cell comprises OLIG2, SOX9 and NKX6.2. In some embodiments, a pluripotent stem cell comprises OLIG2, SOX9 and OCT4. In some embodiments, a pluripotent stem cell comprises OLIG2, NKX6.1 and NKX6.2. In some embodiments, a pluripotent stem cell comprises OLIG2, NKX6.1 and OCT4. In some embodiments, a pluripotent stem cell comprises OLIG2, NKX6.2 and OCT4. In some embodiments, a pluripotent stem cell comprises SOX9, NKX6.1 and NKX6.2. In some embodiments, a pluripotent stem cell comprises SOX9, NKX6.1 and OCT4. In some embodiments, a pluripotent stem cell comprises NKX6.1, NKX6.2 and OCT.

In some embodiments, a pluripotent stem cell comprises at least two transcription factors selected from OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4. In some embodiments, a pluripotent stem cell comprises at least three transcription factors selected from OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4. In some embodiments, a pluripotent stem cell comprises at least four transcription factors selected from OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4. In some embodiments, a pluripotent stem cell comprises OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4.

Stem cells of the present disclosure are engineered. Engineered cells are cells that comprise at least one engineered (e.g., recombinant or synthetic) nucleic acid, or are otherwise modified such that they are structurally and/or functionally distinct from their naturally-occurring counterparts. Thus, a cell that contains an exogenous nucleic acid sequence is considered an engineered cell.

In some embodiments, an engineered cell of the present disclosure is an engineered pluripotent stem cell (e.g., an induced pluripotent stem cell), an oligodendrocyte progenitor cell (OPC), or an oligodendrocyte. As used herein unless indicated otherwise, a cell that is engineered to express a (at least one) transcription factor may be engineered to constitutively express the transcription factor or engineered to inducibly express the transcription factor.

OLIG Transcription Factors

In some embodiments, pluripotent stem cells engineered to produce OPCs comprise an OLIG transcription factor. OLIG transcription factors belong to the basic helix-loop-helix family of transcription factors. Examples of OLIG transcription factors include but are not limited to OLIG1, OLIG2, OLIG3 and OLIG4. In some embodiments, the OLIG transcription factor used in TF-mediate reprogramming of the present disclosure is OLIG1. In some embodiments, the OLIG transcription factor is OLIG2. In some embodiments, the OLIG transcription factor is OLIG3. In some embodiments, the OLIG transcription factor is OLIG4. An OLIG transcription factor, or homolog or variant thereof, as used herein, may be a human or other mammalian OLIG transcription factor. Other OLIG2 transcription factors (e.g., from other species) and other OLIG transcription factors, generally, are known and nucleic acids encoding OLIG transcription factors can be found in publically available gene databases, such as GenBank. In some embodiments, the nucleic acid encoding wild-type human OLIG2 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to the open reading frame of the nucleic acid described in the NCBI Reference Sequence database (RefSeq) under accession number NM_005806. In some embodiments, a nucleic acid encoding OLIG2 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 4. In some embodiments, an amino acid sequence encoding OLIG2 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 9.

SOX Transcription Factors

In some embodiments, pluripotent stem cells engineered to produce OPCs comprise an SOX transcription factor. Members of the SOX (SRY-related HMG-box) family of transcription factors are characterized by a high mobility group (HMG)-box DNA sequence. This HMG box is a DNA binding domain that is highly conserved throughout eukaryotic species. The Sox family has no singular function, and many members possess the ability to regulate several different aspects of development. Examples of SOX transcription factors include but are not limited to SRY, SOX1, SOX2, SOX3, SOX4, SOX5, SOX6, SOX7, SOX8 SOX9, SOX10, SOX11, SOX12, SOX13, SOX14, SOX15, SOX17, SOX18, SOX21 and SOX30. In some embodiments, the SOX transcription factor used in TF-mediate reprogramming of the present disclosure is SRY. In some embodiments, the SOX transcription factor is SOX1. In some embodiments, the SOX transcription factor is SOX2. In some embodiments, the SOX transcription factor is SOX3. In some embodiments, the SOX transcription factor is SOX4. In some embodiments, the SOX transcription factor is SOX5. In some embodiments, the SOX transcription factor is SOX6. In some embodiments, the SOX transcription factor is SOX7. In some embodiments, the SOX transcription factor is SOX8. In some embodiments, the SOX transcription factor is SOX9. In some embodiments, the SOX transcription factor is SOX10. In some embodiments, the SOX transcription factor is SOX11. In some embodiments, the SOX transcription factor is SOX12. In some embodiments, the SOX transcription factor is SOX13. In some embodiments, the SOX transcription factor is SOX14. In some embodiments, the SOX transcription factor is SOX15. In some embodiments, the SOX transcription factor is SOX17. In some embodiments, the SOX transcription factor is SOX18. In some embodiments, the SOX transcription factor is SOX21. In some embodiments, the SOX transcription factor is SOX30. A SOX transcription factor, or homolog or variant thereof, as used herein, may be a human or other mammalian SOX transcription factor. Other SOX transcription factors (e.g., from other species) and other SOX transcription factors, generally, are known and nucleic acids encoding SOX transcription factors can be found in publically available gene databases, such as GenBank. In some embodiments, the nucleic acid encoding wild-type human SOX9 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to the open reading frame of the nucleic acid described in the NCBI RefSeq under accession number Z46629.

In some embodiments, a nucleic acid encoding SOX9 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 2. In some embodiments, an amino acid sequence encoding SOX9 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 7.

NKX Homeobox Transcription Factors

In some embodiments, pluripotent stem cells engineered to produce OPCs comprise a NKX homeobox transcription factor. Members of the NKX family of transcription factors are characterized by a homeobox domain and often regulate development. NKX homeobox transcription factors include members of the NKX2 (NK2) subfamily (e.g., NKX2.1 and NKX2.2) and members of the NKX (NK6) subfamily (e.g., NKX6.1, NKX6.2 and NKX6.3). In some embodiments, the two NKX homeobox transcription factors used in TF-mediate reprogramming of the present disclosure include NKX2.1 and NKX2.2. In some embodiments, the NKX homeobox transcription factor is NKX2.1. In some embodiments, the NKX homeobox transcription factor is NKX2.2. In some embodiments, the two NKX homeobox transcription factors are selected from NKX6.1, NKX6.2 and NKX6.3. In some embodiments, the two NKX homeobox transcription factor are NKX6.1 and NKX6.2. In some embodiments, the two NKX homeobox transcription factor are NKX6.2 and NKX6.3. In some embodiments, the two NKX homeobox transcription factor are NKX6.1 and NKX6.3. In some embodiments, the NKX homeobox transcription factor is NKX6.1. In some embodiments, the NKX homeobox transcription factor is NKX6.2. In some embodiments, the NKX homeobox transcription factor is NKX6.3. A NKX homeobox transcription factor, or homolog or variant thereof, as used herein, may be a human or other mammalian SOX transcription factor. Other NKX homeobox transcription factors (e.g., from other species) and other NKX homeobox transcription factors, generally, are known and nucleic acids encoding NKX homeobox transcription factors can be found in publically available gene databases, such as GenBank. In some embodiments, the nucleic acid encoding wild-type human NKX6.1 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to the nucleic acid described in the NCBI RefSeq under accession number NM_006168. In some embodiments, the nucleic acid encoding wild-type human NKX6.2 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to the nucleic acid described in the NCBI RefSeq under accession number NM_177400. In some embodiments, the nucleic acid encoding wild-type human NKX6.2 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to the open reading frame of the nucleic acid described in the NCBI RefSeq under accession number NM_152568.

In some embodiments, a nucleic acid encoding NKX6.1 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 1. In some embodiments, an amino acid sequence encoding NKX6.1 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 6.

In some embodiments, a nucleic acid encoding NKX6.2 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 3. In some embodiments, an amino acid sequence encoding NKX6.2 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 8.

OCT Transcription Factors

In some embodiments, pluripotent stem cells engineered to produce OPCs comprise an OCT transcription factor. OCT transcription factors are characterized by a bipartite DNA binding domain called a POU domain. Examples of OCT transcription factors include but are not limited to OCT1, OCT2, OCT 4 and OCT6. In some embodiments, the OCT transcription factor used in TF-mediate reprogramming of the present disclosure is OCT1. In some embodiments, the OCT transcription factor is OCT2. In some embodiments, the OCT transcription factor is OCT4 (also called OCT3/4). In some embodiments, the OCT transcription factor is OCT6. An OCT transcription factor, or homolog or variant thereof, as used herein, may be a human or other mammalian OCT transcription factor. Other OCT transcription factors (e.g., from other species) and other OCT transcription factors, generally, are known and nucleic acids encoding OCT transcription factors can be found in publically available gene databases, such as GenBank. In some embodiments, the nucleic acid encoding wild-type human OCT4 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to the open reading from of the nucleic acid described in the NCBI RefSeq under accession number NM_002701, NM_203289, NM_001173531, NM_001285986 or NM_001285987. Non-limiting examples of OCT4 variants encompassed herein include POU5F1, transcript variant 1, POU5F1, transcript variant 2, POU5F1, transcript variant 3, POU5F1, transcript variant 4 and POU5F1 transcript variant 5.

In some embodiments, a nucleic acid encoding OCT4 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 5. In some embodiments, an amino acid sequence encoding OCT4 is at least 80% (e.g., at least 85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 10.

The transcription factors described herein (e.g., an OLIG, SOX, NKX and/or OCT transcription factor) may contain one or more amino acid substitutions relative to its wild-type counterpart. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Methods for Producing OPCs

Provided herein are methods for producing OPCs. In some embodiments, the methods comprise introducing into pluripotent stem cells at least one engineered nucleic acid encoding an oligodendrocyte (OLIG) transcription factor, a SRY-box (SOX) transcription factor, two NKX homeobox transcription factors, and an octamer-binding (OCT) transcription factor, and culturing the pluripotent stem cells to produce O4 positive OPCs (see e.g., Examples 1 and 2).

The OPCs of the present disclosure may further comprise an anti-inflammatory cytokine. See, e.g., Opal et al., Chest. 2000 (4):1162-72 and Benveniste et al., Sci STKE. 2007 (416):pe70 for a discussion of anti-inflammatory cytokines. OPCs in the body do not naturally express anti-inflammatory cytokines (Cannella B, Raine CS. Ann. Neurol. 2004 January; 55(1):46-57). For example, the OPCs of the present disclosure may comprise a nucleic acid encoding an anti-inflammatory cytokine. In some embodiments, an anti-inflammatory cytokine reduces the ability of an OPC to activate the immune system (e.g., suppresses T-cell activation). In some embodiments, the level of proinflammatory cytokines (e.g., cytokines that activate the immune system, including IL17 and IFNγ) secreted by a T-cell in the presence and absence of an OPC may be used to determine the ability of an OPC to activate the immune system. In other embodiments, in vitro cell proliferation assays may be used to assess the effect of a cytokine (e.g., IL10 and IFNβ) that is expressed by an engineered OPC on T cell proliferation. Assays known in the art, including an enzyme-linked immunosorbent assay (ELISA) assay, may be used to determine cytokine levels (See Example 3 in the Examples section below). In some embodiments, an OPC harboring an anti-inflammatory cytokine reduces T-cell activation (e.g., as measured by a lower level of secreted proinflammatory cytokines) by at least 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold compared to an OPC not harboring the anti-inflammatory cytokine under the same or substantially conditions.

Suitable anti-inflammatory cytokines for the present disclosure include, but are not limited to, interferons and interleukins. For example, cells may be engineered to express interferon beta (IFNβ) and/or interleukin 10 (IL10). In some embodiments, an engineered OPC expresses/comprises IFNβ (e.g., also referred to herein as IFNβ1). In some embodiments, an engineered OPC expresses/comprises IL10. In some embodiments, an engineered OPC expresses/comprises both IFNβ and IL10. The sequences of the interferons and cytokines may be obtained from publically available databases, including National Center for Biotechnology Information's GenBank. An exemplary interferon beta 1 (IFNβ) sequence is listed under the GenBank Accession Identifier NM_002176. An exemplary IL10 sequence is listed under the GenBank Accession Identifier NM_000572. It should be understood that, in some embodiments, only the open reading frame is used to express the transcription factors and cytokines described herein.

The anti-inflammatory cytokines may be secreted or promote the secretion of another anti-inflammatory cytokine. For example, an anti-inflammatory cytokine may promote the secretion of IL10. In some embodiments, an OPC harboring an anti-inflammatory cytokine secretes at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 1,500-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold more IL10 than a control counterpart not harboring the anti-inflammatory cytokine.

In some embodiments, an OPC harboring an anti-inflammatory cytokine secretes at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 1,500-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold more IFNβ than a control counterpart not harboring the anti-inflammatory cytokine.

In some embodiments, iPSCs are engineered to express IL-10 and SOX9, NKX6.1, OLIG2, NKX6.2, and/or OCT4. In some embodiments, the engineered iPSCs differentiate into OPCs (e.g., O4+ cells) within 1-10 days of induction of expression of IL-10 and SOX9, NKX6.1, OLIG2, NKX6.2, and/or OCT4. In some embodiments, the engineered cells (e.g., iPSCs or OPCs) secrete IL-10. In some embodiments, the engineered cells (e.g., engineered iPSCs or engineered OPCs) secrete IL-10 at a level that is at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 1,500-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold higher than control cells. Control cells may be naturally-occurring iPSCs or OPCs, engineered iPSCs or engineered OPCs that are not specifically modified to express IL-10, or iPSCs or OPCs that are engineered to inducibly express IL-10 but are cultured under conditions that lack an inducing agent (i.e., IL-10 expression is not induced). In some embodiments, the control cell is the same type of cell (e.g., iPSC or OPC) as the engineered cell. In some embodiments, the iPSCs or OPCs secrete IL-10 at a level that is at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold higher than control cells within 1-10 days of induction of expression of IL-10 and SOX9, NKX6.1, OLIG2, NKX6.2, and/or OCT4.

In some embodiments, an engineered cell secretes IL-10 (e.g., steadily/continuously) over the course of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days of culturing (e.g., in the presence of an inducing agent or in the absence of an inducing agent).

In some embodiments, iPSCs or iPSC-derived cells are engineered to express IL10 and/or IFNβ.

In some embodiments, iPSCs are engineered to express IFNβ and SOX9, NKX6.1, OLIG2, NKX6.2, and/or OCT4. In some embodiments, the engineered iPSCs differentiate into OPCs (e.g., O4+ cells) within 1-10 days of induction of expression of IFNβ and SOX9, NKX6.1, OLIG2, NKX6.2, and/or OCT4. In some embodiments, the engineered cells (e.g., engineered iPSCs or engineered OPCs) secrete IL-10. In some embodiments, the engineered cells (e.g., engineered iPSCs or engineered OPCs) secrete IL-10 at a level that is at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 1,500-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold higher than control cells. Control cells include naturally-occurring iPSCs or OPCs, engineered iPSCs or engineered OPCs that are not specifically modified to express IFNβ, or iPSCs or OPCs that are engineered to inducibly express IFNβ, but are cultured under conditions that lack an inducing agent. In some embodiments, the engineered iPSCs or engineered OPCs secrete IL-10 at a level that is at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold higher than control cells within 1-10 days of induction of expression of IFNβ and SOX9, NKX6.1, OLIG2, NKX6.2, and/or OCT4.

In some embodiments, iPSCs are engineered to express IFNβ, IL-10, and SOX9, NKX6.1, OLIG2, NKX6.2, and/or OCT4. In some embodiments, the engineered iPSCs differentiate into OPCs (e.g., O4+ cells) within 1-10 days of induction of expression of IFNβ, IL-10, and SOX9, NKX6.1, OLIG2, NKX6.2, and/or OCT4. In some embodiments, the engineered iPSCs or engineered OPCs secrete IL-10. In some embodiments, the engineered cells (e.g., engineered OPCs or engineered iPSCs) secrete IL-10 at a level that is at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 1,500-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold higher than control cells. Control cells include naturally-occurring iPSCs or OPCs, engineered iPSCs or engineered OPCs that are not specifically modified to express IFNβ and IL-10, and iPSCs or OPCs that are engineered to inducibly express IFNβ and IL-10, but are cultured under conditions that lack an inducing agent. In some embodiments, the engineered iPSCs or engineered OPCs secrete IL-10 at a level that is at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold higher than control cells within 1-10 days of induction of expression of IFNβ, IL-10, and SOX9, NKX6.1, OLIG2, NKX6.2, and/or OCT4.

A nucleic acid, generally, is at least two nucleotides covalently linked together, and in some instances, may contain phosphodiester bonds (e.g., a phosphodiester “backbone”). A nucleic acid is considered “engineered” if it does not occur in nature. Examples of engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. In some embodiments, an engineered nucleic acid encodes an OLIG (e.g., OLIG2) transcription factor. In some embodiments, an engineered nucleic acid encodes an SOX (e.g., SOX9) transcription factor. In some embodiments, an engineered nucleic acid encodes an NKX homeobox (e.g., NKX6.1 and/or NKX 6.2) transcription factor. In some embodiments, an engineered nucleic acid encodes an OCT (e.g., OCT4) transcription factor. In some embodiments, a single transcription factor is encoded by a single nucleic acid, while in other embodiments, a single nucleic acid may encode two or more transcription factors (e.g., each operably linked to a different promoter, or both operably linked to the same promoter).

Nucleic acids encoding the transcription factors described herein may be introduced into a pluripotent stem cell using any known methods, including but not limited to chemical transfection, viral transduction (e.g. using lentiviral vectors, adenovirus vectors, sendaivirus, and adeno-associated viral vectors) and electroporation. For example, methods that do not require genomic integration include transfection of mRNA encoding one or more of the transcription factors and introduction of episomal plasmids. In some embodiments, the nucleic acids (e.g., mRNA) are delivered to pluripotent stem cells using an episomal vector (e.g., episomal plasmid). In other embodiments, nucleic acids encoding transcriptions factors for reprogramming pluripotent stem cells may be integrated into the genome of the cell. Genomic integration methods are known, any of which may be used herein, including the use of the PIGGYBAC™ transposon system, sleeping beauty system, lentiviral system, adeno-associated virus system, and the CRISPR gene editing system.

In some embodiments, an engineered nucleic acid is present on PIGGYBAC™ transposon vector that comprises PIGGYBAC™ inverted terminal repeat sequences flanking a nucleotide sequence encoding a transcription factor and/or anti-inflammatory cytokine of the present disclosure. PIGGYBAC™ transposases are enzymes that recognize PIGGYBAC™ inverted terminal repeats on each side of an insertion sequence (e.g., sequence encoding a transcription factor of interest), excise the insertion sequence and insert the excised element into another nucleic acid. PIGGYBAC™ transposases may insert excised sequences into target sites with the sequence TTAA. An exemplary sequence encoding PIGGYBAC™ transposase is described in GenBank accession number: EF587698.

In some embodiments, a transcription factor is cloned into a PIGGYBAC™ transposon vector then nucleofected at high copy numbers (e.g., an average of 10 copies per cell) into iPSCs and integrated into the genome by codelivering a PIGGYBAC™ transposase. In some embodiments, a high copy number is 5 to 50 copies, inclusive, (e.g., average of 5 to 50 copies inclusive or exactly 5 to 50 copies inclusive) of a nucleotide sequence encoding a transcription factor (e.g., SOX9, NKX6.1, OLIG2, NKX6.2, and/or OCT4) per cell. In some embodiments, a cell has at least 5, 10, 15, 20, 25, or 50 copies of a nucleotide sequence encoding a transcription factor. In some embodiments, a cell has 15 copies of a nucleotide sequence encoding a transcription factor. In some embodiments, a cell has 5 to 10 copies, 5 to 20 copies, 5 to 30 copies, 5 to 40 copies, 10 to 20 copies, 10 to 30 copies, 10 to 40 copies, 10 to 50 copies, 15 to 20 copies, 15 to 25 copies, 15 to 30 copies, 15 to 35 copies, 15 to 40 copies, 15 to 45 copies, 15 to 50 copies, 20 to 30 copies, 20 to 40 copies, 20 to 50 copies, 30 to 40 copies, 30 to 50 copies, or 40 to 50 copies of a nucleotide sequence encoding a transcription factor. In some instances, the copy number refers to the average copy number of at least one nucleotide sequence encoding at least one transcription factor per cell in a population of cells (e.g., in a polyclonal population of cells). In some instances, the copy number refers to the exact copy number of at least one nucleotide sequence encoding at least transcription factor in a cell or of every cell in a population of cells (e.g., in a clonal population of cells).

In some embodiments, a high copy number may be obtained by introducing a high concentration of DNA into a population of cells. In some embodiments, a high concentration of DNA is a DNA concentration that is greater than or equal to 1,000 ng DNA per 1 million cells, greater than 2,000 ng DNA per 1 million cells, greater than 5,000 ng DNA per 1 million cells, or greater than 10,000 ng DNA per 1 million cells.

In some embodiments, a low copy number is obtained by introducing a low concentration of DNA into a population of cells. In some embodiments, a low concentration of DNA is a DNA concentration that is less than 1,000 ng DNA per 1 million cells, less than 500 ng DNA per 1 million cells, less than 400 ng DNA, less than 300 ng DNA per 1 million cells.

The plasmid may be designed to be, for example, antibiotic resistant and/or inducible (e.g., doxycycline-inducible) in order to permit the selection of transcription factor-integrated cells and/or to control transcription.

In some embodiments, a PIGGYBAC™ transposon vector comprises PIGGYBAC™ inverted terminal repeat sequences flanking a nucleotide sequence encoding an OLIG (e.g., OLIG2) transcription factor. In some embodiments, a PIGGYBAC™ transposon vector comprises PIGGYBAC™ inverted terminal repeat sequences flanking a nucleotide sequence encoding a SOX (e.g., SOX9) transcription factor. In some embodiments, a PIGGYBAC™ transposon vector comprises PIGGYBAC™ inverted terminal repeat sequences flanking a nucleotide sequence encoding a NKX homeobox (e.g., NKX6.1 and/or NKX 6.2) transcription factor. In some embodiments, a PIGGYBAC™ transposon vector comprises PIGGYBAC™ inverted terminal repeat sequences flanking a nucleotide sequence encoding OCT (e.g., OCT4) transcription factor. In some embodiments, the nucleotide sequence encoding any three or more of OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4 are placed, in the same cassette flanked by PIGGYBAC™ inverted terminal repeat sequences. The transcription factors in this cassette may be separated or not separated such that they produce unconnected proteins, for instance by separating the transcription factors by internal ribosome entry sites (IRES) or polypeptide cleavage signals such as 2A sequences. In some embodiments, the nucleotide sequence encoding IFNβ and/or IL10 are flanked by PIGGYBAC™ inverted terminal repeat sequences.

In some embodiments, an engineered nucleic acid is present on an expression plasmid, which is introduced into pluripotent stem cells. In some embodiments, the expression plasmid comprises a selection marker, such as an antibiotic resistance gene (e.g., bsd, neo, hygB, pac, ble, or Sh bla) or a gene encoding a fluorescent protein (RFP, BFP, YFP, or GFP). In some embodiments, an antibiotic resistance gene encodes a puromycin resistance gene. In some embodiments, the selection marker enables selection of cells expressing a transcription factor of interest (e.g., OLIG, SOX, NKX, and/or OCT).

Any of the engineered nucleic acids described herein may be generated using conventional methods. For example, recombinant or synthetic technology may be used to generate nucleic acids encoding the transcription factors described herein. Conventional cloning techniques may be used to insert a transcription factor of interest into a PIGGYBAC™ transposon vector.

In some embodiments, an engineered nucleic acid (optionally present on an expression plasmid) comprises a nucleotide sequence encoding a transcription factor operably linked to a promoter (promoter sequence). In some embodiments, the promoter is an inducible promoter (e.g., comprising a tetracycline-regulated sequence). Inducible promoters enable, for example, temporal and/or spatial control of transcription factor expression.

A promoter control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, activatable, repressible, tissue-specific or any combination thereof. A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. Herein, a promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and/or expression of that sequence.

An inducible promoter is one that is characterized by initiating or enhancing transcriptional activity when in the presence of, influenced by or contacted by an inducing agent. An inducing agent may be endogenous or a normally exogenous condition, compound or protein that contacts an engineered nucleic acid in such a way as to be active in inducing transcriptional activity from the inducible promoter.

Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

A preparation of pluripotent stem cells (e.g., expressing the transcription factor combination as provided herein) may be cultured under standard stem cell culture conditions. For example, the pluripotent stem cells may be cultured in any commercially-available feeder-free maintenance medium for human ESCs and iPSCs, such as mTeSR™1 media. In some embodiments, the pluripotent stem cells are cultured in commercially-available stem cell media without added nutrients or growth factors.

A preparation of pluripotent stem cells (e.g., expressing the transcription factor combination as provided herein) may be cultured, in some embodiments, for as few as 4 to 10 day before producing O4-positive OPCs. In some embodiments, the pluripotent stem cells are cultured for 4-10 days, 4-9 days, 4-8 days, 4-7 days, 4-6 days or 4-5 days. In some embodiments, the pluripotent stem cells are cultured for less than 4 days (e.g., 1, 2 and 3 days) before producing O4-positive OPCs. In some embodiments, at least 10% (at least 20%, 30%, 40%, 50%, 60%, 70% or 80%) of the cells of the preparation express O4 after only 4-10 days (e.g., 4, 5, 6, 7, 8, 9 or 10) of culture.

A preparation of pluripotent stem cells (e.g., expressing the transcription factor combination as provided herein) may include, for example, 10⁴ to 10¹⁰ cells. In some embodiments, a preparation of pluripotent stem cells includes 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ cells.

Expression of O4 and other OPC-specific markers may be assessed based on protein expression or nucleic acid expression using known methods. Additional OPC-specific markers include, but are not limited to, Sox1, Pax6, Nestin, Islet1, A2B5, Sox10, Olig2, Olig1, PDGFRa, NG2, RIP, O1, PLP1, CNPase, GalC, MBP, MAG and MOG. In some embodiments, an OPC does not express OCT4, Nanog, and/or SOX2. Exemplary methods include immunofluorescence using an anti-O4 antibody conjugated to a fluorophore, using western blot analysis with an anti-O4 antibody, quantitative polymerase chain reaction with primers targeting O4, and fluorescence activated cell sorting (FACS) with an anti-O4 antibody. In some embodiments, the methods further comprise sorting for O4-positive OPCs (e.g., using FACS).

In some embodiments, the methods further comprise culturing OPCs to produce oligodendrocytes. Culturing of OPCs may include the use of media comprising factors that induce oligodendrocyte differentiation, including growth hormones (e.g., fibroblast growth factors). Oligodendrocytes may be characterized by cell surface markers (e.g., MOG and GalC). Oligodendrocytes may also, or may alternatively, be characterized by their ability to form myelin. As exemplified below, cells engineered by the methods disclosed herein may be co-cultured with iPSC-derived neurons (e.g., in a polyethylene glycol mold) for a period of time (e.g., a few weeks) and assessed for myelination (e.g., the co-cultures can be fixed, embedded in resin, sectioned, stained and imaged using transmission electron microscopy to assess myelination). In some embodiments, the engineered cells of the present disclosure increase the number of myelinated axons by at least two-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold. In some embodiments, the extent of myelination may be calculated by determining the g-ratio. The axonal g-ratio is the ratio between the inner and the outer diameter of the myelin sheath. In some embodiments, the axonal g-ratio from a subject who has been administered any of the engineered cells of the present disclosure is similar to the g-ratio of myelin from a control (e.g., the subject prior to administration of the engineered cells or a healthy subject). In some embodiments, the axonal g-ratio from a subject who has been administered any of the engineered cells of the present disclosure is similar to the axonal g-ratio from a control (e.g., the subject prior to administration of the engineered cells or a healthy subject) if the axonal g-ratio of myelin from the subject is on average between 0.6-0.8. See, e.g., Mohammadi et al., Front Neurosci. 2015 Nov. 27; 9:441.

In some embodiments, the OPCs described herein have been engineered to express IL-10 and/or IFNβ. In some embodiments, the OPCs are cultured to express IL-10 and/or IFNβ (e.g., with an inducing agent if cytokine expression is controlled by an inducible promoter). Without being bound by a particular theory, OPCs engineered to express IL-10 and/or IFNβ may be useful in the treatment of a subject with a myelin degenerative disease (e.g., Multiple Sclerosis). Without being bound by a particular theory, immune-tolerant cells such as engineered OPCs with capability to secrete IL10 and IFNβ secretion not only can regenerate and remyelinate the axons can protect itself and adjacent cells from the immune attack. In some embodiments, OPCs engineered to secrete IL10 and IFNβ may reduce or suppress the immune response of a host to administered OPCs and promote regeneration.

In some embodiments, the engineered OPCs are cultured with other cells (e.g., neurons) to produce a myelinated organoid. In some embodiments, the myelinated organoid is transplanted into a subject in need thereof (e.g., a subject with a demyelinating disorder). In some embodiments, the organoid is a cerebral organoid (e.g., partial or complete cerebral organoid). In some embodiments, the engineered myelinated organoid is used to compound screening (e.g., therapeutic drugs) or disease modeling.

In some embodiments, a pluripotent stem cell (e.g. iPSC) and/or OPC comprises at least three transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1), (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4). In some embodiments, a pluripotent stem cell (e.g. iPSC) and/or OPC comprises OCT4, SOX9, and NKX6.1. In some embodiments, a pluripotent stem cell (e.g. iPSC) and/or OPC comprises OCT4, OLIG2, and NKX6.1. In some embodiments, a pluripotent stem cell (e.g. iPSC) and/or OPC comprises OCT4, NKX6.2, and NKX6.1. In some embodiments, a pluripotent stem cell (e.g. iPSC) and/or OPC comprises OCT4, SOX9, and OLIG2. In some embodiments, a pluripotent stem cell (e.g. iPSC) and/or OPC comprises OLIG2, SOX9, and NKX6.1.

In some embodiments, a pluripotent stem cell (e.g. iPSC) and/or OPC comprises at least four transcription factors selected from (a) OLIG2, (b) SOX9, (c) NKX6.1, (d) NKX6.2, and (e) OCT4. In some embodiments, a pluripotent stem cell (e.g. iPSC) and/or OPC comprises OCT4, OLIG2, NKX6.1, and NKX6.2. In some embodiments, a pluripotent stem cell (e.g. iPSC) and/or OPC comprises SOX9, OLIG2, NKX6.1, and NKX6.2. In some embodiments, a pluripotent stem cell (e.g. iPSC) and/or OPC comprises OCT4, OLIG2, NKX6.1, and NKX6.2. In some embodiments, a pluripotent stem cell (e.g. iPSC) and/or OPC comprises OCT4, OLIG2, NKX6.1, and SOX9. In some embodiments, a pluripotent stem cell (e.g. iPSC) and/or OPC comprises OCT4, SOX9, NKX6.1, and NKX6.2.

In some embodiments, a pluripotent stem cell (e.g. iPSC) and/or OPC comprises OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4.

Pharmaceutical Compositions and Uses Thereof

Also provided herein are pharmaceutical composition comprising pluripotent stem cells (e.g., induced pluripotent stem cells), OPCs and/or oligodendrocytes produced by any of the methods disclosed herein. The pharmaceutical compositions may further comprise a pharmaceutically-acceptable carrier (e.g., a nanocarrier) or excipient. Hydrogels may also be used as pharmaceutically-acceptable carriers. Non-limiting examples of pharmaceutically-acceptable excipients include water, saline, dextrose, glycerol, ethanol and combinations thereof. The pharmaceutically-acceptable excipient may comprise phosphate buffered saline, a bicarbonate solution, a preservative, a stabilizing agent, an emulsifier (e.g., a phospholipid emulsifier), a solubilizing agent (e.g., surfactant), or a binding agent. The excipient may be selected on the basis of the mode and route of administration, and standard pharmaceutical practice.

General considerations in the formulation and/or manufacture of pharmaceutical agents, such as compositions comprising any of the engineered cells disclosed herein, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

Any of the pharmaceutical compositions disclosed herein may be administered to a subject (e.g., a human subject). Additional exemplary subjects include, but are not limited to, mice, rats, rabbits, horses, dogs, cats, goats, sheep and other animals. The subject may have a demyelinating disorder. Demyelinating disorders include disorders in which myelin surrounding axons is lost or damaged. Magnetic resonance imaging (MRI) may be used to diagnose a demyelinating disorder. Examples of demyelinating disorders include disorders in which myelin in the central nervous system is damaged. In some embodiments, the demyelinating disorder includes damage to OPCs. An exemplary demyelinating disorder is multiple sclerosis.

The term “an effective amount” or a “therapeutically effective amount” as used herein refers to the amount of OPCs and/or oligodendrocytes required to confer therapeutic effect on a subject, either alone or in combination with at least one other active agent. Effective amounts vary, as recognized by those skilled in the art, depending on the route of administration, excipient usage, and co-usage with other active agents. The quantity to be administered depends on the subject to be treated, including, for example, the strength of an individual's immune system or genetic predispositions. Suitable dosage ranges are readily determinable by one skilled in the art and may be on the order of micrograms of the polypeptide of this disclosure. The dosage of the preparations disclosed herein may depend on the route of administration and varies according to the size of the subject.

Suitable routes of administration include, for example, parenteral routes such as intravenous, intrathecal, parenchymal, or intraventricular routes. Suitable routes of administration include, for example, parenteral routes such as intravenous, intrathecal, parenchymal, or intraventricular injection.

EXAMPLES Example 1: Identification of Transcription Factor Combinations Capable of Differentiating Induced Pluripotent Stem Cells (iPSCs) into Oligodendrocyte Progenitor Cells (OPCs)

To identify transcription factor (TF) combinations capable of inducing the formation of OPCs, sixteen different pools of transcription factors were generated and tested for their ability to form OPCs. Each TF was cloned into a PIGGYBAC™ transposon vector (PBAN) and nucleofected at high copy numbers into iPSCs and integrated into the genome with the co-delivery of PIGGYBAC™ transposase. The PBAN plasmid was designed to be puromycin resistant and doxycycline-inducible to enable selection of TF-integrated cells and to express the TFs on demand, respectively. PIGGYBAC™ transposon vectors were used to increase the copy number of the TFs integrated into the genome and to improve the viability of the iPSCs. After cells were nucleofected with transcription factors, cells were treated with puromycin to select for integrants. TF expression was induced by the addition of doxycycline (500 ng/mL) to stem cell media (mTeSR1) for four days (FIG. 1).

Cells expressing each TF combination were then tested for their ability to form OPCs. iPSCs were nucleofected with the TFs and cultured in stem cell media (mTeSR1) without antibiotics and on a Matrigel-coated plate. The cells were kept on Y27632 ROCK inhibitor for one day. After the cells reached 80% confluency puromycin (1 μg/ml) was added to the media to select for integrants. TF expression was induced by addition of doxycycline (500 ng/mL) to stem cell media (mTeSR1) for four days. The cells then were harvested using TrypLE Express (1%) and were fixed in 4% paraformaldehyde. After blocking with 5% BSA, the cells were stained for anti-O4 antibody and the percentage of them that were expressing the O4 marker was assessed using flow cytometry. This study identifies combinations of TFs sufficient to promote OPC formation, without nutrient, growth factor or microenvironmental optimization.

The study identified OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4 as a combination of transcription factors capable of forming OPCs. While iPSCs nucleofected with OLIG2, NKX6.1, NKX6.2, SOX9, and OCT4 constructs continued to form iPSC colonies in the absence of doxycycline (no transcription factor induction), induction of this combination of transcription factors changed the morphology of the iPSCs (FIG. 2). Doxycycline treatment of iPSCs nucleofected with OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4 constructs resulted in cells breaking away from the iPSC colonies and displaying a branched morphology (FIG. 2). These morphological changes are consistent with iPSCs differentiating into OPCs.

Immunohistochemistry analysis was used to determine whether the combination of OLIG2, SOX9, NKX6.1, and OCT4 induced the development of OPCs. iPSCs nucleofected with this combination of TFs were treated with doxycycline to induce expression of the TFs. Immunohistochemistry analysis was then performed with anti-O4, anti-Myelin oligodendrocyte glycoprotein (MOG), anti-NG2 (an integral membrane chondroitin sulfate proteoglycan) and anti-Galactocerebroside (anti-GalC) antibodies to determine the stage of oligodendrocyte development. NG2 was used as early stage OPC marker, O4 was used as an intermediate proliferative premitotic OPC marker, GalC was used as a marker for late stage OPC (post-mitotic) and early mature oligodendrocytes and MOG was used as a marker for mature oligodendrocytes. At four days after induction of TF expression, cells stained positive for O4 (FIG. 3A). At eight days after induction of TF expression, the cells stained positive for O4 (FIG. 3B) and the myelin marker MOG (FIG. 3C). FIGS. 3A-3C demonstrate that O4-expressing O4+ cells can be generated using a subset (OLIG2, SOX9, NKX6.1 and OCT4) of the five transcription factors identified.

Fluorescence activated cell sorting (FACS) was also used for quantitative analysis of conversion percentage of iPSCs to the oligodendrocyte lineage, the cells (engineered by expressing OLIG2, SOX9, NKX6.1, NKX6.2 and OCT4) were stained for anti-NG2, anti-O4, and anti-Galactocerebroside (GalC) surface markers and compared against the nucleofected iPSCs with no exposure to doxycycline (no TF induction). As shown in FIG. 4A, four days after induction of TF (OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4) expression about 40% of doxycycline-treated cells were O4-positive and about 30% of cells were NG2-positive. Few doxycycline-treated cells were positive for the late stage OPC (post-mitotic) and early mature oligodendrocyte marker GalC at 4 days. In contrast, nucleofected iPSCs had negligible expression of all three surface markers in the absence of doxycycline (no induction of TF expression). Thus, the combination of OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4 transcription factors can efficiently induce the development of iPSCs into O4-positive OPCs. As shown in the Examples below, other subsets of this combination can also induce the development of iPSCs into O4-positive OPCs.

To assess the ability of the generated OPCs (engineered by expressing OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4) to form myelin-producing oligodendrocytes, the cells were co-cultured with doxycycline inducible iNGN (Neurogenin-mediated) stem cells in a polyethylene glycol mold. The mold was created using photomicropattering and was shaped like a narrow channel (micro-groove) in order to contain the cells. This method lets the neurons align along the channel and allowed the OPCs to integrate with the neurons and form myelin. To make a micro-groove, cells were co-cultured with iPSCs that were previously developed and engineered to form neurons (FIG. 5). A 10% polyethylene glycol (PEG) solution was added to the collagen-coated transwell. A negative mask to form a microgroove (narrow channel) was used and the light-sensitive solution was irradiated for 30 seconds with UV light. Neurons do not grow in PEG unless it has been functionalized or biomolecules are added to promote neuronal growth. Therefore in this example, it acts as a cell restrictive mold which contains neuronal extensions into the micro-grooves leading to a highly fasciculated aligned neuronal outgrowth. Meanwhile the limited space will increase the interaction between the OPC and neuron-generating iPSCs leading to integration of the two cell types and formation of myelin sheaths with dense lines and intermediate lines, confirming the occurrence and abundance of mature myelin layers. This demonstrates that the OPCs have the capacity to differentiate into functional myelin-forming cells.

After four weeks of co-culture of generated OPCs with iNGN cells, the constructs were fixed, embedded in resin, sectioned, stained and imaged using transmission electron microscope (TEM) (FIG. 4B). TEM analysis showed robust myelin formation around the developing neurons. Thus, the OPCs generated by expressing OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4 are capable of forming myelin-producing oligodendrocytes.

Example 2: Identification of Transcription Factor Combination Subsets Capable of Inducing Differentiating of Induced Pluripotent Stem Cells (iPSCs) into Oligodendrocyte Progenitor Cells (OPCs)

A combination of five TFs (SOX9, NKX6.2, NKX6.1, OCT4, and OLIG2) along with subsets of this combination (FIG. 6, bottom chart) were characterized using the gateway-compatible doxycycline-inducible puromycin resistant PIGGYBAC™ transposon system described above. Each combination (designated OPC0 through OPC10) included three, four or five of the TFs. For example, OPC0 included the combination of OLIG2, OCT4, SOX9, NKX6.1, and NKX6.2. OPC1 included the combination of OCT4, SOX9, and NKX6.1. OPC2 included the combination of OCT4, OLIG2, and NKX6.1. OPC3 included the combination of OCT4, NKX6.1 and NKX6.2. OPC4 included the combination of OCT4, SOX9, and OLIG2. OPC5 included the combination of OLIG2, SOX9, and NKX6.1. OPC6 included the combination of OCT4, OLIG2, NKX6.1, and NKX6.2. OPC7 included the combination of OLIG2, SOX9, NKX6.1, and NKX6.2. OPC8 included the combination of OCT4, OLIG2, NKX6.2, and SOX9. OPC9 included the combination of OCT4, OLIG2, NKX6.1 and SOX9. OPC10 tested the combination of OCT4, SOX9, NKX6.1, and NKX6.2.

The TFs were nucleofected at high copy numbers into iPSCs. The cells were maintained in mTeSR1 and TF over-expression was induced with doxycycline for four days with no additional external cues. The percentage of O4-expressing cells was measured by anti-O4 antibody staining followed by flow cytometry. The results showed that OPC0 and a few subsets (OPC1-OPC10) were able to form O4+ cells in an efficient manner (FIG. 6, top graph). This study includes different integration loci and varied expression levels of the TFs in each cell colony. These results demonstrate that OPC0 is the most efficient in inducing differentiation of iPSCs into O4 expressing OPCs (FIG. 6, top graph). OPC10 also efficiently induced differentiation of iPSCs into O4 expressing OPCs (FIG. 6, top graph).

Individual colonies of the two groups with the highest percentage of O4+ cells after 4 days were selected and expanded. Results showed that out of multiple colonies that were assessed, one of the OPC0 colonies (Colony #1) expressed the highest amount of O4 marker (FIG. 7A) and was subsequently analyzed. OPC0-Colony #1 was stained for early, intermediate and late OPC markers and showed the highest expression of O4 marker after 4 days (FIG. 7B). NG2 was used as early stage OPC marker, O4 was used as an intermediate proliferative premitotic OPC marker, and GalC was used as a marker for late stage OPC (post-mitotic) and early mature oligodendrocytes. After 8 days, the cells were also positive for all three markers (FIG. 7B). In addition, cells that were in their final stages of O4 expression were already maturing into GalC expressing cells, a non-proliferative post-mitotic OPC marker (FIG. 7B). These results demonstrate that these cells have the potential to differentiate and maturate.

The transcriptome profile of the O4-expressing OPCs that were sorted through fluorescence-activated cell sorting (FACS) was also assessed. The assessment was performed on O4+ cells derived from the OPC9 cell line. Cells were lysed using TRIzol and RNA was extracted using Direct-zol RNA MiniPrep. Three replicates of no doxycycline control cells and three replicates of O4+ cells were processed in parallel in each set of library preps. 1 μg RNA was used for Poly(A) isolation using the NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra Directional RNA Library Prep Kit from Illumina. To prevent library over-amplification, a portion of the PCR reaction was amplified by quantitative PCR using SYBR Gold Nucleic Acid Statin on a Roche Lightcycler 480. The remaining reaction was amplified using the number of cycles needed to reach mid-log amplification. The libraries were sequenced on an Illumina MiSeq and sequencing reads were aligned to a human transcriptome reference index using the STAR aligner. Statistical testing was performed using DESeq2's differential expression analysis algorithm. Multiple hypothesis testing was corrected using the False Discovery Rate (FDR) method to generate adjusted p-values. Zero values were imputed to one, and then log 2 transformed values were plotted as a heatmap and clustered by similarity (FIG. 8A). Genes were plotted based on their log 2 fold change compared to hiPSCs and were considered as statistically significant if the adjusted p-value was less than 0.05. A graph comparing the mean of normalized counts and Log 2 fold change is shown in FIG. 8B.

The heatmap showed up-regulation of oligodendrocyte-specific lineage genes, such as OLIG1, PLP1, SOX10, and CSPG4 (NG2) and down-regulation of pluripotency markers such as Nanog (FIG. 8A). SOX10 and OLIG1 are among the top 40 up-regulated genes (FIG. 8C). Olig1 and Sox10 synergistically drive MBP transcription into oligodendrocytes. Sox10 is one of the initial TFs that are expressed after specification of the cells to the oligodendrocyte lineage and in CNS it is only expressed in myelin-forming oligodendroglia. These results confirm the specificity of the generated O4+ cells and their commitment to mature into myelin forming oligodendrocytes using the TF-mediated cell engineering with the five-TF set or its subsets.

Example 3: Production of OPCs Overexpressing IL-10, IFNβ, or the Combination of IL-10 and IFNβ

This example describes production and characterization of stem cell-derived oligodendrocyte progenitor cells (OPCs) overexpressing the cytokines IL-10 and IFNβ, alone and in combination. This example demonstrates that overexpression of IL-10 and/or IFNβ, can stimulate secretion of the anti-inflammatory cytokine IL-10 in engineered OPCs.

Induced pluripotent stem cells were co-electroporated with PIGGYBAC™ expression vectors encoding 1) doxycycline-inducible transcription factors (TFs) (SOX9, NKX6.1, OLIG2, and OCT4) that cause the stem cells to differentiate into oligodendrocyte progenitor cells, and 2) doxycycline-inducible cytokines (e.g., IL10 and IFNβ) that thwart the immune response. A schematic of an example workflow from human induced pluripotent stem cells to induced oligodendrocyte progenitor cells that secrete cytokines to reduce immune response and remyelinate axons for therapeutic effect is provided in FIG. 9. Human IL10 and IFNβ were obtained as ORF clones from the Human ORFeome collection (ORFeome Collaboration, Nat. Meth. 2016; 13(3):191-192). IL10 and IFNβ were expressed from separate constructs. Puromycin was used to select cells that have successfully integrated these vectors into the genome. Then, differentiation and cytokine release were simultaneously induced with the addition of doxycycline. At different time points, the ability of the cells to secrete the cytokines was assayed by enzyme-linked immunosorbent assay (ELISA).

Three different stem cell lines, all with TFs that induce oligodendrocyte differentiation, were generated. In the first line, induced pluripotent stem cells were co-electroporated with the transcription factors and only IL10 (FIG. 10). In the second line, induced pluripotent stem cells were co-electroporated with the transcription factors and only IFNβ (FIG. 11). In the third line, induced pluripotent stem cells were co-electroporated with the transcription factors and both IL10 and IFNβ (FIG. 12).

To determine whether overexpression of IL10 and IFNβ, alone and in combination, could suppress an immune response to engineered OPCs, the levels of secreted IL10 was measured by ELISA assay for each cell line. As shown in FIG. 10, doxycycline treatment induced IL10 secretion in cells co-electroporated with the transcription factors and only IL10. After four days of doxycycline induction, more than 100 μg/μl of secreted IL10 was detected by ELISA assay (FIG. 10). IL10 secretion was observed only in the presence of doxycycline treatment, demonstrating that the induction of IL10 secretion was doxycycline-dependent.

Similarly, as shown in FIG. 11, doxycycline treatment also induced IL10 secretion in cells co-electroporated with the transcription factors and IFNβ. After four days of doxycycline induction, more than 140 μg/μl of secreted IL10 was detected by ELISA assay (FIG. 11). Since IFNβ is upstream of IL10, it is likely that IFNβ secretion promotes IL10 secretion in these cells.

Notably, initial secretion of IL10 was detected as early as 3 days after doxycycline induction in cells co-electroporated with the TFs and both IFNβ and IL10 (FIG. 12). IL10 secretion in these cells peak after 5 days of doxycycline treatment.

Cytokine-releasing OPCs were also generated to achieve a higher (e.g., 10×) IL-10 secretion level. Cells were engineered to inducibly express SOX9, NKX6.1, OLIG2, NKX6.2, and OCT4 and to inducibly express cytokines (IL-10 or IL-10 and IFNβ). ELISA showed that secretion reached levels that are 1000-fold higher than control cells (uninduced). A high amount of IL10 secretion from day 3 that persisted to 7 days of doxycycline induction was observed (FIG. 19A). IL10 secretion was not observed in the absence of doxycycline induction. IL10 was also detected when OPCs were engineered to secrete both IL10 and IFN-beta (FIG. 19B).

These results suggest that overexpression of IFNβ and/or IL10 can induce IL10 secretion in stem cell-derived cells (e.g., engineered OPCs), which can then be used to inhibit T-cell reactivity and remyelinate axons for therapeutic effect. For example, while OPCs expressing the autoantigen MBP elicit an immune response in patients with multiple sclerosis, engineered OPCs overexpressing IL-10 and IFNβ would likely be able to suppress an immune response.

Example 4: Vectors for Stable Overexpression of IL10 and IFNβ

In order to achieve stable overexpression of IL10 and IFNβ, the two constructs may be inserted into PBAN (FIG. 13A). Human IL10 and IFNβ may be obtained as ORF clones from the Human ORFeome collection (ORFeome Collaboration, Nat. Meth. 2016; 13(3):191-192) from Marc Vidal's laboratory (Dana Farber Cancer Institute). To ensure cells express both genes, IL-10 and IFNβ may be stitched together into the same expression unit (IL10_IFNβ) into one vector and then electroporated into stem cells. To select for transfected cells with IL10_IFNβ and TF orthogonally, two selection mechanisms may be applied to the constructs. As the TFs are expressed on a puromycin-resistant vector, IL10_IFNβ constructs may be assembled into a PBAN_GFP vector with GFP as a selection marker. IL10_IFNβ may then be inserted into PBAN_GFP using a Gibson reaction. The cells can then be transfected in order to generate a stable cell line with overexpression of IL-10 and IFNβ simultaneously which may be confirmed by ELISA assay as described above. A functional assay involving T cell proliferation can be performed to assess the extent that IL-10 and IFNβ secretion prevents T cell activation.

As a non-limiting example, a PBAN vector comprising the elements shown in FIG. 13B was used to overexpress IL10 and IFNβ.

Example 5: Effect of Copy Number on Efficiency of OPC Production

To assess the efficiency of OPC production, two amounts of DNA (high and low) were transfected into hiPSCs, and O4 expression (a marker of oligodendrocyte progenitors) was studied after DNA integration, with or without TF over-expression. The cells were engineered to inducibly express OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4 (colonies are from OPC0). High amount of DNA includes concentrations greater than 1000 ng per 1 million cells. Low amount of DNA includes concentrations less than 1000 ng per 1 million cells. To identify the best performing clones, individual colonies, which were generated by either mechanically dislodging individual colonies or sorting single cells into individual wells, were isolated and studied.

As shown herein, transfecting high amounts of DNA increased the number of colonies with high O4 expression (FIG. 14A—high DNA transfected) compared to transfecting low amounts (FIG. 14G—low DNA transfected). To ensure the differentiated cells remained pre-mitotic—an important cell state for engraftment in vivo—GalC expression (a post-mitotic marker) after induction was measured (FIG. 14B). Ideal colonies should show high O4 expression (FIG. 14A) and low GalC expression (FIG. 14B) after doxycycline induction. Without being bound by a particular theory, the increased efficiency of OPC differentiation using a high amount of transfected DNA may be attributed to an increase in the number of copies of integrated TFs in the genome: cells transfected with high amount of DNA showed more TFs (e.g., ˜10-18 copies) integrated into the genome (FIG. 14C) compared to cells transfected with a lower amount of DNA (e.g., ˜4-5 copies) (FIG. 14E). A more detailed analysis for O4 and GalC for the best clones are shown in FIG. 14D (high DNA amount) and FIG. 14F (low DNA amount).

Example 6: Further In Vitro Functional Studies of Engineered OPCs

Next, it was demonstrated that the engineered OPCs were functional. An in vitro assay, where the engineered OPCs were co-cultured with hiPSC-induced neurons, was used. After 3 days, OPCs contacting the neurites of induced neurons were observed (FIG. 15A). In FIG. 15A, OPCs from OPC0 colony 1, which was engineered to inducibly express OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4, was used. To assess whether the engineered OPCs could form myelin sheath in vitro, this co-culture was continued for a total of four weeks. Then, the cells were fixed, a cross-section was obtained, and electron microscopy was performed. Robust myelin formation around axons was observed, demonstrating functional myelination in vitro (FIG. 15B).

To assess the purity of the programmed OPCs, single cell RNA-Seq using oil-droplet emulsions on thousands of single cells was conducted using 10× Genomics' Chromium single cell RNA-seq kit according to manufacturer's instructions. t-SNE (t-distributed stochastic neighbor embedding) plot analysis of CNP, PLP1, and NES expression was conducted. A two-dimensional plot was generated for each gene (CNP, PLP1, and NES) and each point in each plot represented a single cell's transcriptome projected. A single, coherent population of cells, with no sub-populations, was observed, suggesting the programming process was highly pure. It was found that oligodendrocyte-specific markers, such as PLP1 and CNP, were up-regulated as a relatively pure population concordant with the down-regulation of pluripotency genes in the majority of the cell population after four days of induction (data not shown). FIG. 20 is a histogram showing OCT4 expression at the single cell level of cells from OPC0, colony 1, which were engineered to inducibly express OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4. The rightmost peak are cells that have not been treated with doxycycline and therefore remain OCT4-positive stem cells. The leftmost peak and peak with no OCT4 expression (shaded) are cells treated with doxycycline after four days.

This data provides strong evidence that the OPCs programmed within four days with no additional soluble factors are bona fide OPCs.

Example 7: Use of Engineered OPCs to Myelinate Human Organoids

To determine whether the programmed OPCs could form myelin in in vivo-like conditions, human organoids were used. The OPCs were from OPC0, colony 1, which were engineered to inducibly express OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4. To further improve the clinical translation of these programmed OPCs, the human cerebral organoid model was used to evaluate these cells' ability to myelinate human organoids, which feature complex spatial organization and an ensemble of cell types found in human brains. Unmodified hiPSCs were mixed with hiPSCs that could be induced to differentiate into OPCs upon doxycycline addition. After the cerebral organoids were formed without doxycycline, and doxycycline was added to induce differentiation of OPCs in the subset of engineered hiPSCs. After 8 weeks, the human cerebral organoids were sectioned and stained for myelin basic protein (MBP) and for myelin oligodendrocyte glycoprotein (MOG). MOG expression was observed in organoids treated with doxycycline to induce OPC differentiation, but not in organoids cultured without doxycycline (FIGS. 16A-16B). MBP expression was also observed in organoids treated with doxycycline to induce OPC differentiation (FIG. 16C). This in vivo-like human model demonstrates the ability of programmed OPCs to myelinate human neurons.

Example 8: Administration of Engineered OPCs Improved Motor Function in the Shiverer Mouse Model

To determine the myelin-forming ability of the engineered OPCs in vivo, the cells were transplanted into the MBP-deficient Shi/Shi Shiverer mouse model. The OPCs were from OPC0, colony 1, which were engineered to inducibly express OLIG2, SOX9, NKX6.1, NKX6.2, and OCT4. The Shiverer mouse has multiple exon deletions in its MBP gene, leading to minimal MBP protein expression and subsequently a lack of myelin production. This model is the standard animal model that is used to confirm myelin generation in vivo. As these mouse models lack myelin, the mouse models were utilized as an assessment of the myelin forming abilities of our cells in vivo. Any expression of exogenous MBP (or other myelin markers) after cell injection in animals demonstrates that the injected cells promote of myelin formation and are functional. Homozygous Shiverer mice were housed under appropriate conditions with food and water provided and were crossed with heterozygous mice under standard IACUC protocols. Newborn mice (postnatal day=0-3) were cryoanesthesized and 5×10⁴ in 2 μl PBS of cells were injected intracranially using freehand injection while a control group (postnatal day=0-3) received PBS injection.

To perform a behavioral test of motor function, a hind limb suspension test was performed 10 weeks after injection. The mice were suspended by their hind limbs from the lip of a standard 50 ml glass beaker and the posture adopted is scored according to the following criteria: score of 4 indicates normal hind-limb separation with tail raised; score of 3, weakness is apparent and hind limbs closer together but seldom touching each other; score of 2, hind limbs are close to each other and often touching; score of 1, weakness apparent and the hind limbs are almost always in a clasped position with the tail raised; a score of 0 indicates constant clasping of the hind limbs with the tail lowered or failure to hold onto the tube.

The control-untreated healthy mouse had a hind limb suspension score of 4 and the control-untreated Shiverer mice had a lower score of 3 (data not shown). Notably, a Shiverer mouse injected with OPCs had a score of 4, which is the same as the healthy control-untreated mice (data not shown). Thus, the hind limb suspension test demonstrated that the treated Shiverer mouse received a better clinical score 10 weeks after injection compared to the untreated Shiverer mice.

To show the improvement in physiological score was due to myelin growth in a mouse treated with the engineered OPCs, mouse brains were sectioned and stained for myelin. OPC-injected mice stained positive for myelin (FIG. 17B). For reference, healthy mice (fully stained, FIG. 17A) and non-injected mice are shown (no staining, FIG. 17C). The number of myelinated axons was also quantified using transmission electron microscopy (TEM) images. These images show an increase in the number of myelinated axons (FIG. 18B) compared to PBS controls (FIG. 18A). This is quantified in FIG. 18C. The G-ratio quantifies the amount of myelin, with lower numbers showing thicker myelin. FIG. 18D shows the distribution of G-ratio's similar to those found in vivo. The axonal g-ratio range was on average between 0.6 and 0.8.

Example 9: Administration of Engineered OPCs to an Experimental Autoimmune Encephalomyelitis (EAE) Model of Multiple Sclerosis

An experimental autoimmune encephalomyelitis model (EAE) was developed. The EAE model is a standard model for human inflammatory demyelinating diseases such as multiple sclerosis. This model has similar manifestations of the disease such as inflammation, gliosis, and axonal and myelin loss.

In the study described herein, mice were acclimated to the facility for at least 7 days prior to the start of the study. All mice were the same age within 1 week. EAE will be induced in all mice as follows:

Day 0, Hour 0: Immunization with MOG35-55/CFA Day 0, Hour 2: Injection of pertussis toxin Day 1, Hour 0: 2nd injection of pertussis toxin (24 hours after initial immunization)

Mice were injected subcutaneously at two sites in the back with the emulsion component (containing MOG35-55) of Hooke Kit™ MOG35-55/CFA Emulsion PTX, catalog number EK-2110 (Hooke Laboratories, Lawrence Mass.). One site of injection was done in the upper back, approximately 1 cm caudal of the neck line. The second site was in the lower back, approximately 2 cm cranial of the base of the tail. The injection volume was 0.1 mL at each site. Within 2 hours of the injection of emulsion, and then again 24 hours after the injection of emulsion, the pertussis toxin component of the kit was administered intraperitoneally. After disease induction, mice were assigned to groups in a balanced manner to achieve similar average weight across the groups at the start of the study (Day −2).

Survival rate, weight loss and both cumulative and maximum score of each animal will be recorded.

Table 1 below gives the treatment administered to each group. In Table 1, Dox indicates doxycycline and i.v. indicates intravenous administration of indicated engineered OPCs. Twelve (12) animals were used in each group. Test cells A are iPSCs that are capable of expressing SOX9, OLIG2, NKX6.1, NKX6.2, OCT4, IL10, and IFN-beta in the presence of doxycycline.

TABLE 1 Diet and Treatment Regimen of EAE mice. Start of Day of Group Diet diet Treatment Dose Volume treatment Route Purpose 1 Dox Day −2 Vehicle — 150 Day 0, Day 7 i.v. Negative μL/mouse control 2 Dox Day −2 Test cells A 2M 150 Day 7 i.v. Test cells/ms μL/mouse

To study the effect of the cocktail-iPSCs (engineered to inducibly express SOX9, OLIG2, NKX6.1, NKX6.2, OCT4, IL10, and IFN-beta), mice were intravenously (i.v.) injected with cocktail-iPSCs (2×10⁶ cell/mouse) into mice seven days post EAE induction while animals were kept on a doxycycline diet throughout the study. The weight loss and disease progression were monitored in both groups (the vehicle and injected group). The results of the adoptive transfer EAE mouse model with or without intravenous injection of hiPSC-derived OPCs engineered to secrete both IL10 and IFNβ are shown in FIGS. 21A-21B.

The vehicle demonstrated a progressive increase in clinical score from days 10 while a reduction in the clinical score was observed in mice treated with cocktail-iPSCs (P<0.05) (FIG. 21B).

In the experimental group, 2 mice had no disease presentation and there were 0 deaths. All control mice had significant symptom presentation and one died (maximum score=5). Disease reversal from peak presentation in the experimental group include a 1 point reversal in 2 experimental vs 1 control mice, 0.5 point reversal in 5 experimental vs 2 control mice, and progression in 2 experimental vs 8 control mice. One experimental mouse had stable disease.

Example 10: Non-Limiting Examples of Nucleic Acid and Amino Acid Sequences Encoding Transcription Factors

Non-limiting examples of nucleic acid and amino acid sequences encoding transcription factors are provided below. The nucleic acid sequences provided below were used in the examples described above.

Nucleic acid sequence encoding NKX6.1: (SEQ ID NO: 1) ATGTTAGCGGTGGGGGCAATGGAGGGCACCCGGCAGAGCGCATTCCTGC TCAGCAGCCCTCCCCTGGCCGCCCTGCACAGCATGGCCGAGATGAAGAC CCCGCTGTACCCTGCCGCGTATCCCCCGCTGCCTGCCGGCCCCCCCTCC TCCTCGTCCTCGTCGTCGTCCTCCTCGTCGCCCTCCCCGCCTCTGGGCA CCCACAACCCAGGCGGCCTGAAGCCCCCGGCCACGGGGGGGCTCTCATC CCTCGGCAGCCCCCCGCAGCAGCTCTCGGCCGCCACCCCACACGGCATC AACGATATCCTGAGCCGGCCCTCCATGCCCGTGGCCTCGGGGGCCGCCC TGCCCTCCGCCTCGCCCTCCGGTTCCTCCTCCTCCTCTTCCTCGTCCGC CTCTGCCTCCTCCGCCTCTGCCGCCGCCGCGGCTGCTGCCGCGGCCGCA GCCGCCGCCTCATCCCCGGCGGGGCTGCTGGCCGGACTGCCACGCTTTA GCAGCCTGAGCCCGCCGCCGCCGCCGCCCGGGCTCTACTTCAGCCCCAG CGCCGCGGCCGTGGCCGCCGTGGGCCGGTACCCCAAGCCGCTGGCTGAG CTGCCTGGCCGGACGCCCATCTTCTGGCCCGGAGTGATGCAGAGCCCGC CCTGGAGGGACGCACGCCTGGCCTGTACCCCTCATCAAGGATCCATTTT GTTGGACAAAGACGGGAAGAGAAAACACACGAGACCCACTTTTTCCGGA CAGCAGATCTTCGCCCTGGAGAAGACTTTCGAACAAACAAAATACTTGG CGGGGCCCGAGAGGGCTCGTTTGGCCTATTCGTTGGGGATGACAGAGAG TCAGGTCAAGGTCTGGTTCCAGAACCGCCGGACCAAGTGGAGGAAGAAG CACGCTGCCGAGATGGCCACGGCCAAGAAGAAGCAGGACTCGGAGACAG AGCGCCTCAAGGGGGCCTCGGAGAACGAGGAAGAGGACGACGACTACAA TAAGCCTCTGGATCCCAACTCGGACGACGAGAAAATCACGCAGCTGTTG AAGAAGCACAAGTCCAGCAGCGGCGGCGGCGGCGGCCTCCTACTGCACG CGTCCGAGCCGGAGAGCTCATCC Nucleic acid sequence encoding SOX9: (SEQ ID NO: 2) ATGAATCTCCTGGACCCCTTCATGAAGATGACCGACGAGCAGGAGAAGG GCCTGTCCGGCGCCCCCAGCCCCACCATGTCCGAGGACTCCGCGGGCTC GCCCTGCCCGTCGGGCTCCGGCTCGGACACCGAGAACACGCGGCCCCAG GAGAACACGTTCCCCAAGGGCGAGCCCGATCTGAAGAAGGAGAGCGAGG AGGACAAGTTCCCCGTGTGCATCCGCGAGGCGGTCAGCCAGGTGCTCAA AGGCTACGACTGGACGCTGGTGCCCATGCCGGTGCGCGTCAACGGCTCC AGCAAGAACAAGCCGCACGTCAAGCGGCCCATGAACGCCTTCATGGTGT GGGCGCAGGCGGCGCGCAGGAAGCTCGCGGACCAGTACCCGCACTTGCA CAACGCCGAGCTCAGCAAGACGCTGGGCAAGCTCTGGAGACTTCTGAAC GAGAGCGAGAAGCGGCCCTTCGTGGAGGAGGCGGAGCGGCTGCGCGTGC AGCACAAGAAGGACCACCCGGATTACAAGTACCAGCCGCGGCGGAGGAA GTCGGTGAAGAACGGGCAGGCGGAGGCAGAGGAGGCCACGGAGCAGACG CACATCTCCCCCAACGCCATCTTCAAGGCGCTGCAGGCCGACTCGCCAC ACTCCTCCTCCGGCATGAGCGAGGTGCACTCCCCCGGCGAGCACTCGGG GCAATCCCAGGGCCCACCGACCCCACCCACCACCCCCAAAACCGACGTG CAGCCGGGCAAGGCTGACCTGAAGCGAGAGGGGCGCCCCTTGCCAGAGG GGGGCAGACAGCCCCCTATCGACTTCCGCGACGTGGACATCGGCGAGCT GAGCAGCGACGTCATCTCCAACATCGAGACCTTCGATGTCAACGAGTTT GACCAGTACCTGCCGCCCAACGGCCACCCGGGGGTGCCGGCCACGCACG GCCAGGTCACCTACACGGGCAGCTACGGCATCAGCAGCACCGCGGCCAC CCCGGCGAGCGCGGGCCACGTGTGGATGTCCAAGCAGCAGGCGCCGCCG CCACCCCCGCAGCAGCCCCCACAGGCCCCGCCGGCCCCGCAGGCGCCCC CGCAGCCGCAGGCGGCGCCCCCACAGCAGCCGGCGGCACCCCCGCAGCA GCCACAGGCGCACACGCTGACCACGCTGAGCAGCGAGCCGGGCCAGTCC CAGCGAACGCACATCAAGACGGAGCAGCTGAGCCCCAGCCACTACAGCG AGCAGCAGCAGCACTCGCCCCAACAGATCGCCTACAGCCCCTTCAACCT CCCACACTACAGCCCCTCCTACCCGCCCATCACCCGCTCACAGTACGAC TACACCGACCACCAGAACTCCAGCTCCTACTACAGCCACGCGGCAGGCC AGGGCACCGGCCTCTACTCCACCTTCACCTACATGAACCCCGCTCAGCG CCCCATGTACACCCCCATCGCCGACACCTCTGGGGTCCCTTCCATCCCG CAGACCCACAGCCCCCAGCACTGGGAACAACCCGTCTACACACAGCTCA CTCGACCT Nucleic acid sequence encoding NKX6.2: (SEQ ID NO: 3) ATGGACACTAACCGCCCGGGCGCGTTCGTGCTGAGCAGTGCCCCGCTGG CCGCGCTGCACAACATGGCCGAGATGAAGACGTCGCTGTTCCCCTACGC GCTGCAGGGTCCGGCCGGCTTCAAGGCGCCCGCGCTGGGGGGCCTGGGC GCGCAGCTCCCGCTCGGGACCCCGCACGGCATCAGCGACATCCTGGGCC GGCCCGTGGGCGCGGCGGGCGGGGGCCTCCTGGGGGGGCTGCCCCGGCT CAACGGGCTCGCGTCGTCCGCCGGCGTTTACTTCGGGCCCGCGGCCGCT GTGGCGCGCGGCTACCCCAAGCCCCTGGCCGAGCTGCCGGGGCGCCCGC CCATCTTCTGGCCCGGCGTGGTGCAGGGCGCGCCCTGGAGGGACCCGCG TCTGGCTGGCCCGGCCCCGGCCGGCGGCGTCCTGGACAAGGACGGGAAG AAGAAGCACTCGCGCCCGACCTTCTCGGGCCAGCAGATCTTCGCGCTGG AGAAAACCTTCGAGCAGACCAAGTACCTGGCGGGCCCGGAGCGCGCGCG TCTCGCCTACTCGCTGGGCATGACCGAGAGCCAGGTGAAGGTCTGGTTC CAGAACCGCCGGACCAAGTGGCGCAAGCGGCACGCGGTGGAGATGGCGT CGGCCAAGAAGAAGCAGGACTCGGACGCCGAGAAGCTGAAGGTGGGCGG CTCGGACGCGGAGGACGACGACGAATACAACCGGCCCCTGGACCCCAAC TCGGACGACGAGAAGATCACGCGGCTGCTCAAGAAGCACAAACCCTCGA ACTTGGCGCTGGTCAGCCCGTGCGGCGGCGGCGCGGGGGACGCCTTG Nucleic acid sequence encoding OLIG2: (SEQ ID NO: 4) ATGGACTCGGACGCCAGCCTGGTGTCCAGCCGCCCGTCGTCGCCAGAGC CCGATGACCTTTTTCTGCCGGCCCGGAGTAAGGGCAGCAGCGGCAGCGC CTTCACTGGGGGCACCGTGTCCTCGTCCACCCCGAGTGACTGCCCGCCG GAGCTGAGCGCCGAGCTGCGCGGCGCTATGGGCTCTGCGGGCGCGCATC CTGGGGACAAGCTAGGAGGCAGTGGCTTCAAGTCATCCTCGTCCAGCAC CTCGTCGTCTACGTCGTCGGCGGCTGCGTCGTCCACCAAGAAGGACAAG AAGCAAATGACAGAGCCGGAGCTGCAGCAGCTGCGTCTCAAGATCAACA GCCGCGAGCGCAAGCGCATGCACGACCTCAACATCGCCATGGATGGCCT CCGCGAGGTCATGCCGTACGCACACGGCCCTTCGGTGCGCAAGCTTTCC AAGATCGCCACGCTGCTGCTGGCGCGCAACTACATCCTCATGCTCACCA ACTCGCTGGAGGAGATGAAGCGACTGGTGAGCGAGATCTACGGGGGCCA CCACGCTGGCTTCCACCCGTCGGCCTGCGGCGGCCTGGCGCACTCCGCG CCCCTGCCCGCCGCCACCGCGCACCCGGCAGCAGCAGCGCACGCCGCAC ATCACCCCGCGGTGCACCACCCCATCCTGCCGCCCGCCGCCGCAGCGGC TGCTGCCGCCGCTGCAGCCGCGGCTGTGTCCAGCGCCTCTCTGCCCGGA TCCGGGCTGCCGTCGGTCGGCTCCATCCGTCCACCGCACGGCCTACTCA AGTCTCCGTCTGCTGCCGCGGCCGCCCCGCTGGGGGGCGGGGGCGGCGG CAGTGGGGCGAGCGGGGGCTTCCAGCACTGGGGCGGCATGCCCTGCCCC TGCAGCATGTGCCAGGTGCCGCCGCCGCACCACCACGTGTCGGCTATGG GCGCCGGCAGCCTGCCGCGCCTCACCTCCGACGCCAAG Nucleic acid sequence encoding OCT4: (SEQ ID NO: 5) ATGGCGGGACACCTGGCTTCAGATTTTGCCTTCTCGCCCCCTCCAGGTG GTGGAGGTGATGGGCCAGGGGGGCCGGAGCCGGGCTGGGTTGATCCTCG GACCTGGCTAAGCTTCCAAGGCCCTCCTGGAGGGCCAGGAATCGGGCCG GGGGTTGGGCCAGGCTCTGAGGTGTGGGGGATTCCCCCATGCCCCCCGC CGTATGAGTTCTGTGGGGGGATGGCGTACTGTGGGCCCCAGGTTGGAGT GGGGCTAGTGCCCCAAGGCGGCTTGGAGACCTCTCAGCCTGAGGGCGAA GCAGGAGTCGGGGTGGAGAGCAACTCCGATGGGGCCTCCCCGGAGCCCT GCACCGTCACCCCTGGTGCCGTGAAGCTGGAGAAGGAGAAGCTGGAGCA AAACCCGGAGGAGTCCCAGGACATCAAAGCTCTGCAGAAAGAACTCGAG CAATTTGCCAAGCTCCTGAAGCAGAAGAGGATCACCCTGGGATATACAC AGGCCGATGTGGGGCTCACCCTGGGGGTTCTATTTGGGAAGGTATTCAG CCAAACGACCATCTGCCGCTTTGAGGCTCTGCAGCTTAGCTTCAAGAAC ATGTGTAAGCTGCGGCCCTTGCTGCAGAAGTGGGTGGAGGAAGCTGACA ACAATGAAAATCTTCAGGAGATATGCAAAGCAGAAACCCTCGTGCAGGC CCGAAAGAGAAAGCGAACCAGTATCGAGAACCGAGTGAGAGGCAACCTG GAGAATTTGTTCCTGCAGTGCCCGAAACCCACACTGCAGCAGATCAGCC ACATCGCCCAGCAGCTTGGGCTCGAGAAGGATGTGGTCCGAGTGTGGTT CTGTAACCGGCGCCAGAAGGGCAAGCGATCAAGCAGCGACTATGCACAA CGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGGGGACCAGTGT CCTTTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATGGGAG CCCTCACTTCACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAA GCCTTTCCCCCTGTCTCTGTCACCACTCTGGGCTCTCCCATGCATTCAA ACTGA Amino acid sequence encoding NKX6.1: (SEQ ID NO: 6) MLAVGAMEGTRQSAFLLSSPPLAALHSMAEMKTPLYPAAYPPLPAGPPS SSSSSSSSSSPSPPLGTHNPGGLKPPATGGLSSLGSPPQQLSAATPHGI NDILSRPSMPVASGAALPSASPSGSSSSSSSSASASSASAAAAAAAAAA AAASSPAGLLAGLPRFSSLSPPPPPPGLYFSPSAAAVAAVGRYPKPLAE LPGRTPIFWPGVMQSPPWRDARLACTPHQGS1LLDKDGKRKHTRPTFSG QQIFALEKTFEQTKYLAGPERARLAYSLGMTESQVKVWFQNRRTKWRKK HAAEMATAKKKQDSETERLKGASENEEEDDDYNKPLDPNSDDEKITQLL KKHKSSSGGGGGLLLHASEPESSS Amino acid sequence encoding SOX9: (SEQ ID NO: 7) MNLLDPFMKMTDEQEKGLSGAPSPTMSEDSAGSPCPSGSGSDTENTRPQ ENTFPKGEPDLKKESEEDKFPVCIREAVSQVLKGYDWTLVPMPVRVNGS SKNKPHVKRPMNAFMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLN ESEKRPFVEEAERLRVQHKKDHPDYKYQPRRRKSVKNGQAEAEEATEQT HISPNAIFKALQADSPHSSSGMSEVHSPGEHSGQSQGPPTPPTTPKTDV QPGKADLKREGRPLPEGGRQPPIDFRDVDIGELSSDVISNIETFDVNEF DQYLPPNGHPGVPATHGQVTYTGSYGISSTAATPASAGHVWMSKQQAPP PPPQQPPQAPPAPQAPPQPQAAPPQQPAAPPQQPQAHTLTTLSSEPGQS QRTHIKTEQLSPSHYSEQQQHSPQQIAYSPFNLPHYSPSYPPITRSQYD YTDHQNSSSYYSHAAGQGTGLYSTFTYMNPAQRPMYTPIADTSGVPSIP QTHSPQHWEQPVYTQLTRP Amino acid sequence encoding NKX6.2: (SEQ ID NO: 8) MDTNRPGAFVLSSAPLAALHNMAEMKTSLFPYALQGPAGFKAPALGGLG AQLPLGTPHGISDILGRPVGAAGGGLLGGLPRLNGLASSAGVYFGPAAA VARGYPKPLAELPGRPPIFWPGVVQGAPWRDPRLAGPAPAGGVLDKDGK KKHSRPTFSGQQIFALEKTFEQTKYLAGPERARLAYSLGMTESQVKVWF QNRRTKWRKRHAVEMASAKKKQDSDAEKLKVGGSDAEDDDEYNRPLDPN SDDEKITRLLKKHKPSNLALVSPCGGGAGDAL Amino acid sequence encoding OLIG2: (SEQ ID NO: 9) MDSDASLVSSRPSSPEPDDLFLPARSKGSSGSAFTGGTVSSSTPSDCPP ELSAELRGAMGSAGAHPGDKLGGSGFKSSSSSTSSSTSSAAASSTKKDK KQMTEPELQQLRLKINSRERKRMHDLNIAMDGLREVMPYAHGPSVRKLS KIATLLLARNYILMLTNSLEEMKRLVSEIYGGHHAGFHPSACGGLAHSA PLPAATAHPAAAAHAAHHPAVHHPILPPAAAAAAAAAAAAAVSSASLPG SGLPSVGSIRPPHGLLKSPSAAAAAPLGGGGGGSGASGGFQHWGGMPCP CSMCQVPPPHHHVSAMGAGSLPRLTSDAK Amino acid sequence encoding OCT4: (SEQ ID NO: 10) MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGP GVGPGSEVWGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGE AGVGVESNSDGASPEPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELE QFAKLLKQKRITLGYTQADVGLTLGVLFGKVFSQTTICRFEALQLSFKN MCKLRPLLQKWVEEADNNENLQEICKAETLVQARKRKRTSIENRVRGNL ENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKGKRSSSDYAQ REDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGE AFPPVSVTTLGSPMHSN

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

The terms “about” and “substantially” preceding a numerical value mean±10% of the recited numerical value.

Where a range of values is provided, each value between the upper and lower ends of the range are specifically contemplated and described herein. 

What is claimed is:
 1. A pluripotent stem cell comprising at least four transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1), (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4).
 2. The pluripotent stem cell of claim 1 comprising OCT4, SOX9, NKX6.1, and NKX6.2.
 3. The pluripotent stem cell of claim 1 comprising OLIG2, OCT4, SOX9, NKX6.1, NKX6.2.
 4. The pluripotent stem cell of any one of claims 1-3 further comprising at least one cytokine selected from IL-10 and IFNβ.
 5. The pluripotent stem cell of any one of claims 1-4 further comprising IL-10 and IFNβ.
 6. The pluripotent stem cell of any one of claims 1-5, wherein at least one of the transcription factors and/or at least one of the cytokines is encoded by a nucleotide sequence of an engineered nucleic acid.
 7. The pluripotent stem cell of claim 6, comprising 5 to 50 copies of the nucleotide sequence encoding at least one transcription factor.
 8. The pluripotent stem cell of claim 7, wherein the engineered nucleic acid is a modified engineered nucleic acid.
 9. The pluripotent stem cell of any one of claims 6-8, wherein the engineered nucleic acid comprises DNA or RNA.
 10. The pluripotent stem cell of claim 9, wherein the RNA is messenger (mRNA).
 11. The pluripotent stem cell of any one of claims 6-10, wherein the engineered nucleic acid is operably linked to a promoter.
 12. The pluripotent stem cell of claim 11, wherein the promoter is an inducible promoter.
 13. The pluripotent stem cell of any one of claims 6-12, wherein the engineered nucleic acid is present on a PIGGYBAC™ transposon vector.
 14. The pluripotent stem cell of claim 13, wherein the PIGGYBAC™ transposon vector comprises PIGGYBAC™ inverted terminal repeat sequences flanking at least one nucleotide sequence encoding at least one of the transcription factors.
 15. The pluripotent stem cell of claim 13 or 14 further comprising a PIGGYBAC™ transposase or an engineered nucleic acid encoding a PIGGYBAC™ transposase.
 16. The pluripotent stem cell of any one of claims 1-15, wherein the engineered nucleic acid further encodes a selection marker protein.
 17. The pluripotent stem cell of any one of claims 1-16, wherein the pluripotent stem cell is an induced pluripotent stem cell.
 18. The pluripotent stem cell of any one of claims 1-17, wherein the pluripotent stem cell is a human pluripotent stem cell.
 19. A method comprising: introducing into pluripotent stem cells at least four transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1) (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4), optionally wherein each transcription factor is introduced on an engineered nucleic acid comprising a nucleotide sequence encoding the transcription factor, and optionally wherein at least two transcription factors are encoded by the same engineered nucleic acid; and culturing the pluripotent stem cells to produce a population of cells comprising oligodendrocyte progenitor cells (OPCs).
 20. A method comprising: introducing into pluripotent stem cells at least one engineered nucleic acid encoding IL-10, IFNβ, or a combination thereof; introducing into the pluripotent stem cells at least four transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1) (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4), optionally wherein each transcription factor is introduced on an engineered nucleic acid comprising a nucleotide sequence encoding the transcription factor, and optionally wherein at least two transcription factors are encoded by the same engineered nucleic acid; and optionally culturing the pluripotent stem cells to produce a population of cells comprising oligodendrocyte progenitor cells (OPCs).
 21. The method of claim 19 or 20, comprising introducing 5 to 50 copies of at least one nucleotide sequence encoding at least one transcription factor.
 22. The method of any one of claims 19-21, wherein OCT4, SOX9, NKX6.1, and NKX6.2 are introduced into the pluripotent stem cells.
 23. The method of any one of claims 19-22, wherein at least 40% of the cells of the population express O4.
 24. The method of any one of claim 19-21 or 23, wherein OLIG2, OCT4, SOX9, NKX6.1, NKX6.2 are introduced into the pluripotent stem cells.
 25. The method of any one of claim 19-22 or 24, wherein at least 25% of the cells of the population express O4.
 26. The method of any one of claims 19-25, wherein the cells are cultured for 1 to 10 days.
 27. The method of any one of claims 19-26, wherein at least 25% of the cells of the population express O4 following 4 days of culture.
 28. The method of any one of claim 19 or 21-27, wherein at least one engineered nucleic acid encoding a cytokine selected from IL-10 and IFNβ is introduced into the pluripotent stem cells.
 29. The method of claim 28, wherein at least one engineered nucleic acid encoding IL-10 and at least one engineered nucleic acid encoding IFNβ are introduced into the pluripotent stem cells, optionally wherein IL-10 and IFNβ are encoded by the same engineered nucleic acid.
 30. The method of any one of claims 20-29, wherein the OPCs secrete IL-10.
 31. The method of claim 30, wherein the OPCs secrete IL-10 following 3, 4, 5, or 7 days of culturing.
 32. The method of claim 31, wherein the level of IL-10 secreted by the OPCs is at least 50-fold higher, at least 100-fold, or at least 1000-fold higher than control cells, wherein the control cells are (a) naturally-occurring OPCs, (b) OPCs that are not engineered to express IL-10, and/or (c) OPCs that are engineered to express IL-10 under an inducible promoter and are cultured in the absence of an inducing agent.
 33. The method of any one of claims 19-32, further comprising culturing the OPCs to produce oligodendrocytes.
 34. The method of any one of claims 19-33, wherein at least one engineered nucleic acid is a modified engineered nucleic acid.
 35. The method of any one of claims 19-34, wherein at least one engineered nucleic acid comprises DNA or RNA.
 36. The method of claim 35, wherein the RNA is messenger (mRNA).
 37. The method of any one of claims 19-36, wherein at least one engineered nucleic acid is operably linked to a promoter.
 38. The method of claim 37, wherein the promoter is an inducible promoter.
 39. The method of claim 38, wherein the pluripotent stem cells are cultured in the presence of an agent that induces the inducible promoter to activate transcription of the transcription factors and/or cytokines.
 40. The method of any one of claims 19-39, wherein at least one engineered nucleic acid is present on a PIGGYBAC™ transposon vector.
 41. The method of claim 40, wherein the PIGGYBAC™ transposon vector comprises PIGGYBAC™ inverted terminal repeat sequences flanking at least one nucleotide sequence encoding at least one of the transcription factors.
 42. The method of claim 40 or 41 further comprising a PIGGYBAC™ transposase or an engineered nucleic acid encoding a PIGGYBAC™ transposase.
 43. The method of any one of claims 19-42, wherein at least one engineered nucleic acid further encodes a selection marker protein.
 44. The method of any one of claims 19-43, wherein the pluripotent stem cell is an induced pluripotent stem cell.
 45. The method of any one of claims 19-44, wherein the pluripotent stem cell is a human pluripotent stem cell.
 46. A pharmaceutical composition comprising OPCs produced by the method of any one of claims 19-45.
 47. A pharmaceutical composition comprising oligodendrocytes produced by the method of any one of claims 33-45.
 48. A method comprising administering to a subject having a demyelinating disorder the pharmaceutical composition of claim 46 or
 47. 49. The method of claim 48, wherein the demyelinating disorder is multiple sclerosis.
 50. A pluripotent stem cell or an oligodendrocyte progenitor cell comprising OCT4, OLIG2, NKX6.1, and SOX9.
 51. A pluripotent stem cell or an oligodendrocyte progenitor cell comprising OCT4, OLIG2, and SOX9.
 52. A pluripotent stem cell or an oligodendrocyte progenitor cell comprising OLIG2, NKX6.1, and SOX9.
 53. The pluripotent stem cell or an oligodendrocyte progenitor cell of any one of claims 50-52 further comprising IL-10.
 54. The pluripotent stem cell or an oligodendrocyte progenitor cell of claim 53 further comprising IFNβ.
 55. A pluripotent stem cell engineered to express IL-10 and/or IFNβ, optionally wherein the pluripotent stem cell is an induced pluripotent stem cell.
 56. An oligodendrocyte progenitor cell engineered to express IL-10 and/or IFNβ.
 57. The cell of claim 55 or 56, wherein the cell is engineered to express IL-10 and IFNβ.
 58. The cell of any one of claims 55-57, wherein the IL-10 and/or IFNβ are encoded by at least one nucleic acid that is stably integrated into the genome of the cell.
 59. The cell of any one of claims 55-58, wherein the cell secretes at least 50-fold, 100-fold, or 1000-fold more IL-10 than a control.
 60. The cell of claim 59, wherein the control cell is (a) a naturally-occurring pluripotent stem cell or naturally-occurring OPC, (b) a pluripotent stem cell or OPC that is not engineered to express IL-10, IFNβ, or a combination thereof, or (c) a pluripotent stem cell or OPC that is engineered to express IL-10, IFNβ, or a combination thereof under an inducible promoter and is cultured under conditions that lack an inducible agent.
 61. A method comprising: introducing into pluripotent stem cells at least four transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1) (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4), optionally wherein each transcription factor is introduced on an engineered nucleic acid comprising a nucleotide sequence encoding the transcription factor, and optionally wherein at least two transcription factors are encoded by the same engineered nucleic acid; culturing the pluripotent stem cells to produce a population of cells comprising oligodendrocyte progenitor cells (OPCs); and culturing the OPCs, optionally with another cell type, to produce a myelinated organoid.
 62. The method of claim 61, further comprising transplanting the myelinated organoid into a subject in need thereof.
 63. A method comprising: introducing into pluripotent stem cells at least four transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1) (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4), optionally wherein each transcription factor is introduced on an engineered nucleic acid comprising a nucleotide sequence encoding the transcription factor, and optionally wherein at least two transcription factors are encoded by the same engineered nucleic acid to produce engineered pluripotent stem cells capable of expressing at least four transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1) (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4).
 64. The method of claim 63 further comprising administering the engineered pluripotent stem cells, or OPCs derived from the pluripotent stem cells, to a subject in need thereof.
 65. The method of any one of claims 63-64 comprising inducing expression of the at least four transcription factors.
 66. A pluripotent stem cell comprising at least three transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1), (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4).
 67. The pluripotent stem cell of claim 67 comprising OCT4, SOX9, and OLIG2.
 68. The pluripotent stem cell of claim 67 comprising OCT4, SOX9, and NKX6.1. 